Protease inhibitor conjugates and antibodies useful in immunoassay

ABSTRACT

Activated haptens useful for generating immunogens to HIV protease inhibitors, immunogens useful for producing antibodies to HIV protease inhibitors, and antibodies and labeled conjugates useful in immunoassays for the HIV protease inhibitor saquinavir. The novel haptens feature an activated functionality at the central, non-terminal hydroxyl group. Also described are monoclonal antibodies specific for saquinavir having less than 10% cross-reactivity with lopinavir, nelfinavir, amprenavir, ritonavir, and indinavir, and a murine hybridoma producing said antibodies.

RELATED APPLICATIONS

This application divisional of U.S. Ser. No. 10/669,831, filed Sep. 24,2003 now U.S. Pat No. 7,193,065, which is a continuation-in-part of U.S.patent application Ser. No. 10/192,052 filed Jul. 10, 2002, nowabandoned which claims priority to U.S. Provisional Application No.60/305,192 filed Jul. 13, 2001.

FIELD OF THE INVENTION

This invention relates to novel protease inhibitor conjugates andantibodies useful in immunoassay. More specifically, this inventionrelates to novel activated haptens useful for generating immunogens toHIV protease inhibitors, to novel immunogens useful for producingantibodies to HIV protease inhibitors, and to novel antibodies andlabeled conjugates useful in immunoassays for HIV protease inhibitors.

BACKGROUND OF THE INVENTION

HIV protease inhibitors are an important new class of drugs which havemade a significant impact on the health care of AIDS patients since thefirst one, saquinavir, was introduced to the marketplace in 1995.Examples of other protease inhibitors include amprenavir, indinavir,nelfinavir, lopinavir, ritonavir, and atazanavir. They are especiallyeffective in combination with other anti-HIV drugs such as reversetranscriptase inhibitors or with other HIV protease inhibitors. In spiteof remarkable success with these new therapeutic regimens, there arestrong indications that results would be much improved if therapeuticdrug testing methods were available for monitoring the concentrations ofprotease inhibitors. Not all patients respond optimally to proteaseinhibitor combination therapies. Even those who do respond cansubsequently develop drug resistance due to the notoriously high rate ofmutation of the HIV virus. However, it has been shown that there is aclear relationship between plasma levels of the protease inhibitors andtherapeutic efficacy based upon decreased viral load and increased CD4cell count. One problem lies in the fact that the drugs are metabolizedextensively and are subject to complex drug-drug interactions. Theresults are extremely complex pharmacokinetics and a strong element ofunpredictability between dosage and resultant drug levels at anyparticular time for any particular patient. With therapeutic drugmonitoring, drug dosages could be individualized to the patient, and thechances of keeping the virus in check would be much higher. But routinetherapeutic drug monitoring of protease inhibitors would require theavailability of simple automated tests adaptable to high throughputclinical analyzers. Currently most reports on therapeutic drugmonitoring of protease inhibitors have used HPLC methods which are slow,labor-intensive, and expensive. Recently there was a report of aradioimmunoassay (RIA) method for saquinavir (Wiltshire et al.,Analytical Biochemistry 281, 105-114, 2000). However, such a methodwould not be adaptable to high-throughput therapeutic drug monitoringand, like all RIA methods, suffers from the disadvantages of havingregulatory, safety and waste disposal issues related to the radioactiveisotope label used in the assay. The most desirable assay formats fortherapeutic drug monitoring are non-isotopic immunoassays, and suchmethods have heretofore been unknown for monitoring HIV proteaseinhibitors.

As indicated above, HPLC has been the method of choice for monitoringHIV protease inhibitors. Two recent reports in the literature describeHPLC assays for the simultaneous determination of several proteaseinhibitors in human plasma, Poirier et al., Therapeutic Drug Monitoring22, 465-473, 2000 and Remmel et al., Clinical Chemistry 46, 73-81, 2000.

Chemical and biological assays generally involve contacting the analyteof interest with a pre-determined amount of one or more assay reagents,measuring one or more properties of a resulting product (the detectionproduct), and correlating the measured value with the amount of analytepresent in the original sample, typically by using a relationshipdetermined from standard or calibration samples containing known amountsof analyte of interest in the range expected for the sample to betested. Typically, the detection product incorporates one or moredetectable labels which are provided by one or more assay reagents.Examples of commonly used labels include functionalized microparticles,radioactive isotope labels such as ¹²⁵I and ³²P, enzymes such asperoxidase and beta-galactosidase and enzyme substrate labels,fluorescent labels such as fluoresceins and rhodamines, electron-spinresonance labels such as nitroxide free radicals, immunoreactive labelssuch as antibodies and antigens, labels which are one member of abinding pair such as biotin-avidin and biotin-streptavidin, andelectrochemiluminescent labels such as those containing a rutheniumbipyridyl moiety. Sandwich assays typically involve forming a complex inwhich the analyte of interest is sandwiched between one assay reagentwhich is ultimately used for separation, e.g., antibody, antigen, or onemember of a binding pair, and a second assay reagent which provides adetectable label. Competition assays typically involve a system in whichboth the analyte of interest and an analog of the analyte compete for abinding site on another reagent, e.g., an antibody, wherein one of theanalyte, analog or binding reagent possesses a detectable label.

Copending U.S. patent application Ser. No. 09/712,525 filed Nov. 14,2000 having the same assignee as the present application and publishedas EP 1 207 394 on May 22, 2002, describes a non-isotopic immunoassayfor an HIV protease inhibitor comprising incubating a sample containingthe inhibitor with a receptor specific for the inhibitor or for ametabolite of said inhibitor and further with a conjugate comprising ananalog of the inhibitor and a non-isotropic signal generating moiety.Signal generated as a result of binding of the inhibitor by the receptoris measured and correlated with the presence or amount of proteaseinhibitor in the original sample. The protease inhibitor conjugates ofthe present invention are especially useful in such an assay.

SUMMARY OF THE INVENTION

The present invention relates to novel activated haptens useful forgenerating immunogens to HIV protease inhibitors. These activatedhaptens have the general structure:I—X—(C═Y)_(m)-L-Awherein I is an HIV protease inhibitor radical, X is O or NH, Y is O, Sor NH, m is 0 or 1, L is a linker consisting of from 0 to 40 carbonatoms arranged in a straight chain or a branched chain, saturated orunsaturated, and containing up to two ring structures and 0-20heteroatoms, with the proviso that not more than two heteroatoms may belinked in sequence, and A is an activated functionality selected fromthe group consisting of active esters, isocyanates, isothiocyanates,thiols, imidoesters, anhydrides, maleimides, thiolactones, diazoniumgroups and aldehydes.

The present invention also relates to novel immunogens having thefollowing structure:[I—X—(C═Y)_(m)-L-Z]_(n)—Pwherein I is an HIV protease inhibitor radical, X is O or NH, Y is O, Sor NH, m is 0 or 1, L is a linker comprising 0 to 40 carbon atomsarranged in a straight chain or a branched chain, saturated orunsaturated, and containing up to two ring structures and 0-20heteroatoms, with the proviso that not more than two heteroatoms arelinked in sequence, Z is a moiety selected from the group consisting of—CONH—, —NHCO—, —NHCONH—, —NHCSNH—, —OCONH—, —NHOCO—, —S—, —NH(C═NH)—,—N═N—, —NH—, and

P is a polypeptide, a polysaccharide, or a synthetic polymer, and n is anumber from 1 to 50 per 50 kilodaltons molecular weight of P.

The present invention also relates to novel labeled conjugates havingthe following structure:[I—X—(C═Y)_(m)-L-Z]_(n)-Qwherein I is an HIV protease inhibitor radical, X is O or NH, Y is O, S,or NH, m is 0 or 1, L is a linker comprising 0 to 40 carbon atomsarranged in a straight chain or a branched chain, saturated orunsaturated, and containing up to two ring structures and 0-20heteroatoms, with the proviso that not more than two heteroatoms arelinked in sequence, Z is a moiety selected from the group consisting of—CONH—, —NHCO—, —NHCONH—, —NHCSNH—, —OCONH—, —NHOCO—, —S—, —NH(C═NH)—,—N═N—, —NH—, and

Q is a non-isotopic label, and n is a number from 1 to 50 per 50kilodaltons molecular weight of Q.

The present invention also comprises specific monoclonal antibodies tosaquinavir, nelfinavir, indinavir, amprenavir, lopinavir, and ritonavirhaving less than 10% cross-reactivity to other protease inhibitors.Finally, the present invention comprises antibodies generated from theimmunogens of the invention as well as immunoassay methods and test kitswhich incorporate the antibodies and labeled conjugates of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a scheme for synthesis of O-acylated ritonaviractivated haptens, LPH immunogen and BSA conjugate.

FIG. 2 illustrates a scheme for synthesis of O-acylated saquinaviractivated haptens, KLH immunogen and BSA conjugates.

FIG. 3 illustrates a scheme for synthesis of O-acylated amprenaviractivated haptens, KLH immunogen and BSA conjugate.

FIG. 4 illustrates a scheme for synthesis of O-acylated indinaviractivated haptens, KLH immunogen and BSA conjugate.

FIG. 5 illustrates a scheme for synthesis of O-acylated nelfinaviractivated haptens, KLH immunogen and BSA conjugate.

FIG. 6 illustrates a scheme for synthesis of O-acylated lopinaviractivated haptens, KLH immunogen and BSA conjugate.

FIG. 7 illustrates a scheme for synthesis of an alternative O-acylatedsaquinavir and ritonavir activated haptens and an alternative ritonavirimmunogen.

FIG. 8 illustrates a scheme for synthesis of an N-acylated amprenavirimmunogen.

FIG. 9 illustrates a scheme for synthesis of an O-alkylated nelfinavirimmunogen

FIGS. 10( a) and 10 (b) illustrate a scheme for synthesis ofO-carbamylated saquinavir activated haptens.

FIG. 11 illustrates a scheme for synthesis of O-carbamylated nelfinaviractivated haptens.

FIG. 12 illustrates a scheme for synthesis of O-acylated saquinaviractivated hapten.

FIG. 13 illustrates a scheme for synthesis of O-acylated saquinaviractivated haptens with peptide linkers and maleimide end groups. Alsoillustrated is a KLH immunogen and BSA conjugate derived from the latteractivated haptens.

FIG. 14 illustrates a scheme for synthesis of fluorescein conjugates ofsaquinavir and ritonavir and of a biotin conjugate of indinavir.

FIG. 15 is a chart showing antibody titers generated in Example 77 usingconjugates 2G, 2W, 2D and 2S.

FIG. 16 illustrates the structures of the conjugates used in Example 77.

FIG. 17 are graphs showing the cross-reaction of monoclonal antibody<INDIN> M 1.158.8 and monoclonal antibody <INDIN> M 1.003.12 withindinavir, nelfinavir, ritonavir, saquinavir and amprenavir as describedin Example 80.

FIG. 18 illustrates a scheme for synthesis of O^(ar)-MEMO^(c)-succinimido-oxycarbonylmethyl-nelfinavir ether.

FIG. 19 illustrates a scheme for synthesis ofO^(c)-succinimido-oxycarbonylmethyl-saquinavir ether.

FIG. 20 is a graph showing the cross-reaction of monoclonal antibody<AMPREN> M 1.1.52 with indinavir, saquinavir, ritonavir, lopinavir, andnelfinavir as described in Example 81.

FIG. 21 is a graph showing the cross-reaction of monoclonal antibody<LOPIN> M 1.1.85 with indinavir, saquinavir, ritonavir, lopinavir, andnelfinavir as described in Example 82.

FIG. 22 is a graph showing the cross-reaction of monoclonal antibody<RITON> M 1.5.44 with indinavir, saquinavir, amprenavir, lopinavir, andnelfinavir as described in Example 83.

FIG. 23 illustrates a scheme for synthesis of O-acylated atazanaviractivated haptens, KLH immunogen, and BSA conjugate.

Throughout the specification, numbers in boldface type are used to referto chemical structures illustrated in the drawings.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, analyte refers to a substance, or group of substances,whose presence or amount thereof is to be determined.

Antibody means a specific binding partner of the analyte and is anysubstance, or group of substances, which has a specific binding affinityfor the analyte to the essential exclusion of other unrelatedsubstances. The term includes polyclonal antibodies, monoclonalantibodies and antibody fragments.

Haptens are partial or incomplete antigens. They are protein-freesubstances, mostly low molecular weight substances, which are notcapable of stimulating antibody formation, but which do react withantibodies. The latter are formed by coupling a hapten to a highmolecular weight carrier and injecting this coupled product into humansor animals. Examples of haptens include therapeutic drugs such asdigoxin and theophylline, drugs of abuse such as morphine and LSD,antibiotics such as gentamicin and vancomycin, hormones such as estrogenand progesterone, vitamins such as vitamin B12 and folic acid, thyroxin,histamine, serotonin, adrenaline and others.

An activated hapten refers to a hapten derivative that has been providedwith an available site for reaction, such as by the attachment of, orfurnishing of, an activated group for synthesizing a derivativeconjugate.

The term linker refers to a chemical moiety that connects a hapten to acarrier, immunogen, label, tracer or another linker. Linkers may bestraight or branched, saturated or unsaturated carbon chains. They mayalso include one or more heteroatoms within the chain or at termini ofthe chain. By heteroatoms is meant atoms other than carbon which areselected from the group consisting of oxygen, nitrogen and sulfur. Theuse of a linker may or may not be advantageous or needed, depending onthe specific hapten and carrier pairs.

A carrier, as the term is used herein, is an immunogenic substance,commonly a protein, that can join with a hapten, thereby enabling thehapten to stimulate an immune response. Carrier substances includeproteins, glycoproteins, complex polysaccharides and nucleic acids thatare recognized as foreign and thereby elicit an immunologic responsefrom the host.

The terms immunogen and immunogenic as used herein refer to substancescapable of producing or generating an immune response in an organism.

The terms conjugate and derivative refer to a chemical compound ormolecule made from a parent compound or molecule by one or more chemicalreactions.

As used herein, a detector molecule, label or tracer is an identifyingtag which, when attached to a carrier substance or molecule, can be usedto detect an analyte. A label may be attached to its carrier substancedirectly or indirectly by means of a linking or bridging moiety.Examples of labels include enzymes such as β-galactosidase andperoxidase, fluorescent compounds such as rhodamine and fluoresceinisothiocyanate (FITC), luminescent compounds such as dioxetanes andluciferin, and radioactive isotopes such as ¹²⁵I.

The term active ester within the sense of the present inventionencompasses activated ester groups which can react with nucleophilessuch as, but not limited to, free amino groups of peptides,polyaminoacids, polysaccharides or labels under such conditions that nointerfering side reactions with other reactive groups of thenucleophile-carrying substance can usefully occur.

An object of the present invention is to provide novel activated haptensthat can be used to generate immunogens to HIV protease inhibitors.These activated haptens take the general structure:I—X—(C═Y)_(w)-L-Awherein I is an HIV protease inhibitor radical, X is O or NH, Y is O, S,or NH, m is 0 or 1, L is a linker consisting of from 0 to 40 carbonatoms arranged in a straight chain or a branched chain, saturated orunsaturated, and containing up to two ring structures and 0-20heteroatoms, with the proviso that not more than two heteroatoms may belinked in sequence, and A is an activated functionality selected fromthe group consisting of active esters, isocyanates, isothiocyanates,thiols, imidoesters, anhydrides, maleimides, thiolactones, diazoniumgroups and aldehydes.

As used herein, an HIV protease inhibitor radical is the intact druglacking only a hydroxyl group or an amino group, XH, where X is O or NH.The X and C═Y moieties include, but are not limited to, esters (where Xis O, Y is O, and m is 1), amides (where X is NH, Y is O, and m is 1),urethanes (where X is O, Y is O, m is 1, and the first atom in Ladjacent to C═Y is N), ureas (where X is NH, Y is O, m is 1, and thefirst atom in L adjacent to C═Y is N), thioureas (where X is NH, Y is S,m is 1, and the first atom in L adjacent to C═Y is N), amidines (where Xis NH, Y is NH, and m is 1), ethers (where X is O, and m is 0) andamines (where X is NR wherein R is H or lower alkyl, and m is 0). “Loweralkyl” means methyl, ethyl, propyl and isopropyl groups. Preferredactivated haptens are esters or urethanes formed with the central,non-terminal hydroxyl group common to all HIV protease inhibitors. Thiscentral hydroxyl group is functionally important for the therapeuticactivity of the protease inhibitors but also provides a convenienthandle for derivatization and linker attachment. Moreover, generally themetabolism of the protease inhibitors takes place at terminal residues,and therefore the central hydroxyl groups are attractive sites forimmunogens designed to generate antibodies which discriminate betweenparent drug and metabolites. As used herein, this central hydroxyl groupis designated as HO^(c). When the hydrogen of the central hydroxyl groupis replaced by a (C═Y)_(m)-L-A group, the residual bonded oxygen isshown as O^(c).

The linker L serves the purpose of providing an additional spacerbetween the terminal activated functionality A and the HIV proteaseinhibitor radical, the first spacer being the X and C═Y groups. Linkerlength and composition are well known to those skilled in the art tohave important effects on immunogen response and conjugate performance.There are many examples of commercially available or easily synthesizedlinkers in the literature for attachment to hydroxyl and amino groups.For a good treatise on this subject, the reader is referred toBioconjugate Techniques, G. Hermanson, Academic Press, 1996. In somecases the additional linker L is dispensed with the C═Y moiety isdirectly attached to an activated functionality A. An example of apreferred linker moiety L is —(CH₂)_(x)—NH— where x is 1-12.Particularly preferred is x=5 in combination with C═Y where Y is O(i.e., aminocaproyl esters). Such linkers are formed by acylation of anHIV protease inhibitor with an N-protected amino acid (i.e.,aminocaproic acid). The protecting group is preferably one which isremoved under mildly basic or acidic conditions so as not to affect theintegrity of the X—C═Y bonds or other moieties in the HIV proteaseinhibitor radical. An example of an N-protecting group removed undermildly basic conditions is fluorenylmethyloxycarbonyl (FMOC). An exampleof an N-protecting group easily removed with acid is t-butyloxycarbonyl(BOC). Many other suitable N-protecting groups are well known in the art(see “Protective Groups” in Organic Synthesis, 2nd edition, T. Greeneand P. Wuts, Wiley-Interscience, 1991).

The acylation reaction of HIV protease inhibitor hydroxyl or aminogroups with N-protected amino acids is accomplished by usingcondensation reagents such as carbodiimides with or without a catalyst.A preferred combination is dicyclohexylcarbodiimide withdimethylaminopyridine as catalyst. The acylation reaction is carried outin a suitable solvent such as methylene chloride at 0-35° C. for a timewhich typically ranges from 0.5 to 7 days. Following isolation of theproduct, the N-protecting group is removed. For the preferred FMOCprotecting group, this is accomplished by treatment with a solution of10% piperidine in methylene chloride for 0.5 to 2 hours. The amino groupof the resultant aminoacyl-protease inhibitor is amenable to acylationreactions with a wide variety of carboxyl activated linker extensions orlabels which are well known to those skilled in the art to which thepresent invention belongs. Linker extension is often performed at thisstage to generate terminal activated groups A such as active esters,isocyanates and maleimides. For example, reaction of theaminoacyl-protease inhibitor with one end of homobifunctionalN-hydroxysuccinimide esters of bis-carboxylic acids such as terephthalicacid will generate stable N-hydroxysuccinimide ester terminated linkeradducts which are useful for conjugation to amines on polypeptides,polysaccharides, and labels. Linker extension can also be accomplishedwith heterobifunctional reagents such as maleimido alkanoic acidN-hydroxysuccinimide esters to generate terminal maleimido groups forsubsequent conjugation to thiol groups on polypeptides and labels.Alternatively, an amino-terminated linker can be extended with aheterobifunctional thiolating reagent which reacts to form an amide bondat one end and a free or protected thiol at the other end. Some examplesof thiolating reagents of this type which are well known in the art are2-iminothiolane (2-IT), succinimidyl acetylthiopropionate (SATP) andsuccinimido 2-pyridyldithiopropionate (SPDP). The incipient thiol groupis then available, after deprotection, to form thiol ethers withmaleimido or bromoacetylated modified immunogens or labels. Yet anotheralternative is to convert the amino group of the amino-terminated linkerinto a diazonium group and hence the substance into a diazonium salt,for example, by reaction with an alkali metal nitrite in the presence ofacid, which is then reactive with a suitable nucleophilic moiety, suchas, but not limited to, the tyrosine residues of peptides, proteins,polyaminoacids and the like. Examples of suitable amino-terminatedlinkers for conversion to such diazonium salts include aromatic amines(anilines), but may also include the aminocaproates and similarsubstances referred to above. Such anilines may be obtained bysubstituting into the coupling reaction between the hydroxyl of aprotease inhibitor and an N-protected amino acid, as discussed above,the corresponding amino acid wherein the amino group is comprised of anaromatic amine, that is, an aniline, with the amine suitably protected,for example, as an N-acetyl or N-trifluoroacetyl group, which is thendeprotected using methods well-known in the art. Other suitable amineprecursors to diazonium salts will be suggested to one skilled in theart of organic synthesis.

Another favored type of heterobifunctional linker is a mixed activeester/acid chloride such as succinimido-oxycarbonyl-butyryl chloride.The more reactive acid chloride end of the linker preferentiallyacylates amino or hydroxyl groups on the HIV protease inhibitor to giveN-hydroxysuccinimidyl ester linker adducts directly (see Examples 40 foramprenavir and 8 for ritonavir).

Yet another type of terminal activated group useful in the presentinvention is an aldehyde group. Aldehyde groups may be generated bycoupling the hydroxyl of the protease inhibitor with an alkyl or arylacid substituted at the omega position (the distal end) with a maskedaldehyde group such as an acetal group, such as 1,3-dioxolan-2-yl or1,3-dioxan-2-yl moieties, in a manner similar to that describedpreviously, followed by unmasking of the group using methods well-knownin the art. (See, e.g., T. Greene and P. Wuts, supra). Alternatively,alkyl or aryl carboxylic acids substituted as the omega position with aprotected hydroxy, such as, for example, an acetoxy moiety, may be usedin the coupling reaction, followed by deprotection of the hydroxy andmild oxidation with a reagent such as pyridinium dichromate in asuitable solvent, preferably methylene chloride, to give thecorresponding aldehyde. Other methods of generating aldehyde-terminatedsubstances will be apparent to those skilled in the art.

In certain cases, it is desirable to introduce polarity into the linkercomposition to improve solubility or performance characteristics in theassay of interest. Particularly useful in this regard are peptidelinkers, which offer a wide diversity of possibilities for optimizationand are readily accessible by solid phase peptide synthesis or by othermeans.

Another approach which is particularly useful for generating acylatedHIV protease inhibitors with urethane, urea or thiourea bonds at thepoint of attachment to the protease inhibitor is to react the hydroxylor amino group of the protease inhibitor with a linker isocyanate or alinker isothiocyanate. For example, a carboxyalkylisocyanate with orwithout a protecting group on the carboxyl group may be reacted directlywith the target hydroxyl group on a protease inhibitor to give aprotected carboxyalkylurethane or a carboxyarylurethane. The protectedcarboxy is preferably an ester which is removed under basic or acidicconditions. Once freed, the carboxyl group may be activated to give anactive ester for subsequent conjugation or which may be directlyconjugated to polypeptides, polysaccharides and labels. Alternatively, apreactivated carboxyalkylisocyanate or carboxyarylisocyanate such asN-hydroxysuccinimidyl-isocyanatobenzoate may be reacted directly withprotease inhibitor hydroxyl or amine groups to give linker-acylatedprotease inhibitor with an active ester terminus.

Yet another approach for generating urethane, urea and thiourea bonds atthe point of attachment to the HIV protease inhibitor is to first treatthe target hydroxyl or amine function with phosgene or thiophosgene togive an oxycarbonyl chloride or oxythiocarbonyl chloride. The latterintermediates react readily with amines to give urethanes, ureas orthioureas. Alternative phosgene equivalents such as carbonyldiimidazoleor disuccinimidyl-carbonate will react similarly.

Another approach is also useful for generating alkylated derivatives ofHIV protease inhibitors out of the central hydroxyl group. For example,a protease inhibitor (or properly protected protease inhibitor) can bereacted with a strong base under suitable conditions to deprotonate thecentral hydroxyl group. This can be reacted with a variety of halo alkylreagents bearing a protected carboxylic acid or appropriately protectedfunctionality such as an amino group protected as the phthalimide toform ether linkages. The protected carboxyl group is preferably an esterwhich is removed under acid or basic conditions. The free carboxylicacid group may be activated to give an active ester for subsequentconjugation to polypeptides, polysaccharides and labeling groups. Thefree amino group, after deprotection, can also be extended using abi-functional linker with an activated carboxylic acid group or it canbe coupled to a polypeptide by means of a urea linkage or similar group.

For generation of amidine adducts, the amine of an HIV proteaseinhibitor is reacted with an imidoester, many of which are known inbioconjugate chemistry as linkers (see Hermanson, ibid.)

Alternatively, protease inhibitors derivatized with linkers bearing animidate moiety (imido ester; or iminium group) as the activated groupmay be obtained by, for example, using a linker carrying a suitableprecursor group, for example, a terminal nitrile group, whenappropriately functionalizing a protease inhibitor. For example, anO^(c)-alkylated derivative, or an O^(ar)-alkyl derivative, for example,of nelfinavir, or N^(ar)-alkyl derivative, for example, of amprenavir,carrying a terminal nitrile may be synthesized in a manner analogous tothat described above, followed by conversion of the nitrile to animidate group by methods known in the art, for example, by treatmentwith hydrogen chloride in an alcohol. See also: Hermanson, ibid; andJerry March, Advanced Organic Chemistry, 3^(rd) Ed., John Wiley & sons,1985. Other methods of obtaining imido esters will be suggested to oneskilled in the art.

In certain protease inhibitors with multiple hydroxy groups, i.e.,indinavir and nelfinavir, or hydroxy groups and amino groups in the sameprotease inhibitor, i.e., amprenavir, it may be necessary to protect oneof the groups in order to effect clean reaction at the other functionalgroup. For example, the indinavir indane hydroxyl group can be protectedwith an isopropylidine group bridging to the adjacent amide nitrogen(see compound 4A, Example 4). For the purposes of this application theindane hydroxyl group is labeled as HO^(in) to distinguish it fromHO^(c). The isopropylidine protected indinavir HO^(in) by extension isdesignated as O^(in)N^(in)-isopropylidinyl.

In another example, nelfinavir aromatic hydroxyl (HO^(ar) as usedherein) is protected with a t-butyldimethylsilyl (TBDMS) group beforereaction with the central hydroxyl group, HO^(c) (see compound 5A,Example 5). Nelfinavir aromatic hydroxyl is also protected with amethoxy ethoxymethyl ether (MEM) group (see compound 5M, Example 31).Many other suitable protecting groups for alcohols and phenols are knownin the art, and the reader is again referred to Green and Wuts, ibid.for further examples.

In other cases, adjustment of the reaction conditions will allow forselection of one functional group over another, and protection will notbe needed. An example of the latter approach is the selective acylationof amprenavir hydroxyl group or amino group (see Examples 3 and 40).Another example is the selective alkylation of nelfinavir phenolichydroxyl group (HO^(ar)) in the presence of unprotected aliphaticcentral hydroxyl group (HO^(c), see Example 36).

From the description above, it is evident that there are many variationsof linker technology which will provide an activated terminal group A inthe HIV protease inhibitor hapten compositions of interest. Some ofthese variations will now be described in more detail. Active esters arethe most preferred A group. Active esters of the invention are reactivewith nucleophiles, especially primary amines, at relatively lowtemperatures, generally 0-100° C. in a variety of aqueous andnon-aqueous solvent mixtures. Typical conditions for active estercouplings with primary or secondary amines to give amides are reactionin dipolar aprotic solvents such as N,N-dimethylformamide (DMF) ordimethylsulfoxide (DMSO) with or without added water at roomtemperature. A buffer or a tertiary amine is often added to maintain thebasic pH needed to keep the primary amine reactant in a deprotonatedstate. Typical active esters are p-nitrophenyl esters,N-hydroxysulfosuccinimidyl esters, N-hydroxysuccinimidyl esters,1-hydroxybenzotriazolyl esters and pentafluorophenyl esters. Especiallypreferred are the N-hydroxysuccinimidyl esters because of their balanceof stability, reactivity and the easy removal of side productN-hydroxysuccinimide. Other active esters are well known to thoseskilled in the art and may be used similarly.

An alternative activation method for protease inhibitor linkersterminated with carboxylic acids is in situ preparation of anhydrides.Particularly preferred are the mixed carbonic anhydrides formed withalkylchloroformates such as isobutylchloroformate. These mixedanhydrides are readily formed at temperatures typically ranging from−30° C. to +30° C., usually −20° C. to 0° C., by the reaction ofcarboxylic acid and alkylchloroformate in the presence of a tertiaryamine such as triethylamine or N-methylmorpholine in solvents such asDMF or tetrahydrofuran (THF) for 5 minutes to 1 hour. The mixedanhydride is then reacted with amino groups on labels, immunogens andcarriers, typically for 5 minutes to 1 hour at 0° C. to +30° C. to givestable amide conjugates. Also, symmetrical anhydrides may be formed byreaction of two equivalents of a protease inhibitor linker carboxylicacid group with carbodiimides such as dicyclohexylcarbodiimides (DCC) orethyl-dimethylaminopropyl-carbodiimide (EDAC) in a variety of solventssuch as THF, DMF or dichloromethane. The activation and coupling toamines is typically carried out under similar conditions as the mixedanhydride coupling above.

Yet another activation method for protease inhibitor linkers terminatedwith carboxylic acids is conversion to masked thiol groups, such asthiolactones, by coupling of the carboxylic acid group with a substancesuch as homocysteine thiolactone. (See, e.g., U.S. Pat. No. 5,302,715.)The resulting linker-thiolactone may then be unmasked with mild base togive a terminal thiol which is then reactive with moieties likemaleimido groups or bromoacetyl or iodacetyl groups, such as onmaleimido- or haloacetyl-modified peptides, polysaccharides,polyaminoacids, labels and the like, to give thio-maleimido orthio-acetyl adducts in a similar manner to that described previously.

Other useful A groups are isothiocyanate or isocyanate moieties.Isothiocyanates also react readily with nucleophiles such as primaryamines to give thioureas under conditions similar to the active esterreaction described above, while isocyanates react similarly to giveureas. An added advantage of the isothiocyanate or isocyanate reactionis that it is an addition rather than a substitution, and thereforethere is no side-product to be concerned about as in the case of activeesters. Isocyanate equivalents, such as, for example,p-nitrophenyloxycarbonylamino moieties react similarly with primaryamines to give ureas.

Finally, when the target nucleophile is a thiol group, maleimides areespecially preferred because of their rapid formation of thiol ethersunder very mild conditions, i.e., ambient temperature and neutral pH.Alternatively, active haloalkyl A groups such as iodoacetyl orbromoacetyl also react readily to form stable thiol ethers.

Another object of the invention is to provide novel immunogens with thefollowing structure:[I—X—(C═Y)_(m)-L-Z]_(n)—Pwherein I is an HIV protease inhibitor radical, X is O or NH, Y is O, S,or NH, m is 0 or 1, L is a linker consisting of from 0 to 40 carbonatoms arranged in a straight chain or a branched chain, saturated orunsaturated, and containing up to two ring structures and 0-20heteroatoms, with the proviso that not more than two heteroatoms arelinked in sequence, Z is a moiety selected from the group consisting of—CONH—, —NHCO—, —NHCONH—, —NHCSNH—, —OCONH—, —NHOCO—, —S—, —NH(C═NH)—,—N═N—, —NH—, and

P is a polypeptide, a polysaccharide or a synthetic polymer, and n is anumber from 1 to 50 per 50 kilodaltons molecular weight of P.

For immunogens, the preferred mode of the invention is to link from thecentral hydroxyl group common to all HIV protease inhibitors by anacylation reaction to form an ester bond (i.e., X is O, m is 1 and Y isO). A wide variety of linkers L and activated functionalities A may beused as described above. Thus an activated hapten of the typeI—X—(C═Y)m-L-A is constructed and reacted with an immunogenic carriersubstance. The immunogenic carrier is typically a polypeptide or apolysaccharide with a molecular weight more than 10 kD. Preferredimmunogenic carriers are polypeptides with a molecular weight more than100 kD. Examples of preferred carrier substances are keyhole limpethemocyanin (KLH), Limulus polyphemus hemocyanin (LPH) and bovinethyroglobulin (BTG). The reaction between the activated hapten and aminogroups on the carrier is typically carried out in a buffered mixture ofwater and a water miscible organic solvent such as DMSO at roomtemperature for 0.5 to 5 days. The pH of the buffer is typically between6 and 8 for active esters, isocyanates, and isothiocyanates, or between7 and 10 for imidates, and is adjusted according to the known reactivityof the carrier amino groups and the activated functionality. In the casewhere the terminal group A is a maleimide, the reactive groups on thecarrier are thiols. These thiol groups are either native to the carrieror may be introduced using thiolating reagents such as 2-IT or SATP. Theoptimum pH for the conjugation of maleimides to thiol groups to givethioethers is typically between 5 and 7. Following the reaction, theimmunogen is dialyzed or subjected to size exclusion chromatography inorder to remove unconjugated hapten and organic solvent.

An alternative method of obtaining immunogens is to react an activatedhapten wherein A is aldehyde with the amino groups of a carrier proteinor polypeptide to form a Schiff base, followed by reduction with mildreducing agents such as a cyanoborohydride, to form a stable amine bond.Variations on this last approach will also be suggested to those skilledin the art to which the present invention belongs.

Yet another object of the present invention is to provide antibodies toHIV protease inhibitors generated from the immunogens of the invention.In order to generate antibodies, the immunogen can be prepared forinjection into a host animal by rehydrating lyophilized immunogen toform a solution or suspension of the immunogen. Alternatively, theimmunogen may be used as a previously prepared liquid solution or as asuspension in buffer. The immunogen solution is then combined with anadjuvant such as Freund's to form an immunogen mixture. The immunogenmay be administered in a variety of sites, at several doses, one or moretimes, over many weeks.

Preparation of polyclonal antibodies using the immunogens of theinvention may follow any of the conventional techniques known to thoseskilled in the art. Commonly, a host animal such as a rabbit, goat,mouse, guinea pig, or horse is injected with the immunogen mixture.Further injections are made, with serum being assessed for antibodytiter until it is determined that optimal titer has been reached. Thehost animal is then bled to yield a suitable volume of specificantiserum. Where desirable, purification steps may be taken to removeundesired material such as nonspecific antibodies before the antiserumis considered suitable for use in performing assays.

Monoclonal antibodies may be obtained by hybridizing mouse lymphocytes,from mice immunized as described above, and myeloma cells using apolyethylene glycol method such as the technique described in Methods inEnzymology 73 (Part B), pp. 3-46, 1981.

In the case of ELISA assays, protease inhibitor derivatives coupled tobovine serum albumin (BSA) are preferred for coating of microtiterplates.

Another object of the invention is to provide novel labeled conjugateswith the following structure:[I—X—(C═Y)_(m)-L-Z]_(n)-Qwherein I is an HIV protease inhibitor radical, X is O or NH, Y is O, S,or NH, m is 0 or 1, L is a linker consisting of from 0 to 40 carbonatoms arranged in a straight chain or a branched chain, saturated orunsaturated, and containing up to two ring structures and 0-20heteroatoms, with the proviso that not more than two heteroatoms arelinked in sequence, Z is a moiety selected from the group consisting of—CONH—, —NHCO—, —NHCONH—, —NHCSNH—, —OCONH—, —NHOCO—, —S—, —NH(C═NH)—,—N═N—, —NH—, and

Q is a non-isotopic label, and n is a number from 1 to 50 per 50kilodaltons molecular weight of Q.

For the synthesis of conjugates of HIV protease inhibitors andnon-isotropic labels, similar procedures as for the preparation ofimmunogens are employed.

Alternatively, the activated haptens may be conjugated to amino or thiolgroups on enzymes to prepare labels for ELISA application. Some examplesof useful enzymes for ELISA for which conjugates are well-known in theart are horseradish peroxidase (HRP), alkaline phosphatase andβ-galactosidase. Conjugates of proteins including enzymes are typicallyprepared in a buffered mixture of water and water miscible organicsolvents followed by dialysis analogous to the conditions forpreparation of immunogens. In the case of latex agglutination assays,conjugates with aminated dextran carriers having molecular weightsbetween 10 kD and 300 kD, preferably 40 kD, are especially useful. Theseconjugates are prepared in buffered solvent mixtures as above or in ananhydrous organic solvent such as DMSO containing a tertiary amine suchas triethylamine to promote the reaction. In the case of labels of smallmolecular weight, i.e., less than 1 kD, reaction conditions are adjustedaccording to the nature of the label. One label which is particularlypreferred is biotin in combination with labeled avidin or streptavidin.The versatility of (strept)avidin/biotin systems for non-isotopicdetection is well known in the art of bioconjugate chemistry (seeHermanson, ibid.). A variety of enzyme- and fluorophore-labeledconjugates of avidin and streptavidin are commercially available todetect biotin-labeled substances in a high affinity interaction.Furthermore, a variety of biotinylating agents are commerciallyavailable to react with activated functionalities A. For example, abiotin-amine derivative may be reacted with activated haptens of theinvention in which A is an active ester, isocyanate or isothiocyanate togive biotin amide, urea and thiourea conjugates respectively. Thesecoupling reactions are typically carried out in a dipolar aproticsolvent such as DMF or DMSO containing an organic base such astriethylamine at room temperature for 0.5 to 5 days. The biotinconjugates are preferentially isolated by chromatographic methods suchas reverse phase HPLC.

Other preferred labels are fluorophores such as fluorescein, rhodamine,TEXAS RED fluorescent dye (Molecular Probes, Inc.), dansyl, and cyaninedyes, e.g., Cy-5, of which many activated derivatives are commerciallyavailable. Generally, these conjugates may be prepared similarly asbiotin conjugates in a dipolar aprotic solvent containing a tertiaryamine followed by chromatographic isolation.

It is also possible to use a reporter group as label which is indirectlycoupled to a detection system. One example is biotin as described above.Another example is mycophenolic acid derivatives for inhibition ofinosine monophosphate dehydrogenase as described in PCT publication WO200101135, published Jan. 4, 2001.

It will be obvious to those skilled in the art that there are otherpossibilities for non-isotopic labels including electrochemiluminescentlabels such as ruthenium bipyridyl derivatives, chemiluminescent labelssuch as acridinium esters, electrochemical mediators, and a variety ofmicroparticles and nanoparticles which can be used for the inventionafter suitable introduction of suitable nucleophilic groups on thelabel, e.g., amines or thiols, for reaction with activated groups A onthe HIV protease inhibitor activated hapten.

SPECIFIC EMBODIMENTS

In the examples that follow, numbers in boldface type refer to thecorresponding structure shown in the drawings. These examples arepresented for illustration only without any intent to limit theinvention.

O-Acylation of Protease Inhibitors EXAMPLE 1 Synthesis ofO^(c)-(N-FMOC-aminocaproyl)-ritonavir (1A)

Ritonavir (1, 0.3605 g), FMOC-aminocaproic acid (0.1944 g, AdvancedChemTech, Louisville, Ky.), dimethylaminopyridine (0.0672 g, AldrichChemical Co., Milwaukee, Wis.) and dicyclohexylcarbodiimide (0.1238 g,Fluka Chemical Corp., Milwaukee, Wis.) were stirred overnight inanhydrous methylene chloride (5 mL) at room temperature. The mixture wasfiltered, and the filtrate was evaporated to dryness under reducedpressure and directly purified by silica gel (EM Science Cat. No.9385-9, silica gel 60, 230-400 mesh ASTM) chromatography under apositive pressure of nitrogen (3% methanol in chloroform elution) toyield O^(c)-(N-FMOC-aminocaproyl)-ritonavir (1A) as a white solid(0.5023 g, 95%). M+H 1056.2

EXAMPLE 2 Synthesis of O^(c)-(N-FMOC-aminocaproyl)-saquinavir (2A)

O^(c)-(N-FMOC-aminocaproyl)saquinavir (2A) was prepared from saquinavirmethanesulfonate (2, 0.1917 g) following the conditions described inExample 1, except more methylene chloride (75 mL) was used and thereaction was stirred for 2 days (A. Farese-Di Giorgio et al., AntiviralChem. and Chemother. 11, 97-110, 2000) (0.2354 g, 94%). M+H 1006.2

EXAMPLE 3 Synthesis of O^(c)-(N-FMOC-aminocaproyl)-amprenavir (3A)

O^(c)-(N-FMOC-aminocaproyl)-amprenavir (3A) was prepared from amprenavir(3) (0.1517 g) following the conditions described in Example 1 (0.2248g; 89%). M+H 841

EXAMPLE 4 Synthesis ofO^(c)-(N-FMOC-aminocaproyl)-O^(in),N^(in)-isopropylidinyl-indinavir (4B)

Indinavir sulfate (4, 0.3559 g), camphorsulfonic acid (0.1401 g, AldrichChemical Co.), and magnesium sulfate (4 mg) were refluxed overnight indimethoxypropane (5 mL, A. Farese-Di Giorgio et al, Antiviral Chem. andChemother, 11, 97-110, 2000). The mixture was partitioned betweenmethylene chloride and saturated aqueous sodium bicarbonate. The organiclayer was evaporated to dryness under reduced pressure and directlypurified by silica gel chromatography (4% methanol in chloroformelution) to yield O^(in),N^(in)-isopropylidyl-indinavir (4A) as acolorless oil (0.2350 g; 72%). M+H 654.4.

O^(c)-(N-FMOC-aminocaproyl)-O^(in),N^(in)-isopropylidinyl-indinavir (4B)was prepared from O^(in),N^(in)-isopropylidyl-indinavir (4A, 0.1317 g)following the conditions described in Example 1 (0.1742 g; 87%). M+H989.4

EXAMPLE 5 Synthesis ofO^(c)-N-FMOC-aminocaproyl)-O^(ar)-TBDMS-nelfinavir (5B)

Nelfinavir (5, 0.2839 g) and sodium hydride (18 mg) were stirred in DMF(3 mL) for 15 minutes, t-Butyldimethylsilyl (TBDMS) chloride (0.1130 g)was added and the reaction was stirred overnight. The mixture wasevaporated to dryness under reduced pressure and directly purified bysilica gel chromatography (3% methanol in chloroform elution) to yieldO^(ar)-TBDMS-protected nelfinavir (5A) as a white foam (0.2857 g; 84%).M+H 682.4.

O^(c)-(N-FMOC-aminocaproyl)-O^(ar)-TBDMS-nelfinavir (5B) was preparedfrom O^(ar)-TBDMS protected nelfinavir (5A, 0.3297 g) following theconditions described in Example 1 (0.3385 g; 69%). M+H 1017.7

EXAMPLE 6 Synthesis of O^(c)-(N-FMOC-aminocaproyl)-lopinavir (6A)

O^(c)-(N-FMOC-aminocaproyl)-lopinavir (6A) was prepared from lopinavir(6, 0.712 g) following the conditions described in Example 1 (0.500 g;45%). M+H 964.4

EXAMPLE 7 Synthesis ofO^(c)-[3-(4′-carboxyphenyl)-propionyl)]-saquinavir (2H)

3-(4′-Carboxyphenyl)-propionyl-saquinavir (2H) was prepared fromsaquinavir methanesulfonate (2, 0.1534 g) and3-(4′-carboxyphenyl)-propionic acid (0.0485 g, Lancaster Synthesis Inc.,Windham, N.H.) following the conditions described in Example 1 (0.1041g; 61%). M+H 847.4. Spectral data (¹H-NMR) for the product wascompatible with esterification at the alkyl carboxy rather than the arylcarboxy.

EXAMPLE 8 Synthesis of O^(c)-(succinimido-oxycarbonyl-butyryl)-ritonavir(1G)

Succinimido-oxycarbonyl-butyryl chloride, i.e.,5-(2,5-dioxo-1-pyrrolidinyl-oxy)-5-oxo-pentanoyl chloride, is preparedaccording to Antonian et al., EP 0 503 454. Ritonavir (1, 0.2163 g) andsuccinimido-oxycarbonyl-butyryl chloride (0.0817 g) were stirredovernight in anhydrous DMF (3 mL) at 50° C. The mixture was evaporatedto dryness under reduced pressure and directly purified by silica gelchromatography (30% tetrahydrofuran in ethyl acetate elution) to yieldO^(c)-succinimido-oxycarbonyl-butyryl)-ritonavir (1G) as a white solid(0.1220 g, 44%). M+H 931.8

Deprotection of O-Acylated Protease Inhibitors EXAMPLE 9 Synthesis ofO^(c)-(aminocaproyl)-ritonavir (1B)

O^(c)-(N-FMOC-aminocaproyl)-ritonavir (1A) from Example 1 (0.2113 g) wasstirred 1 hour in 10% piperidine in anhydrous methylene chloride (4 mL)at room temperature. The mixture was evaporated to dryness under reducedpressure and directly purified by silica gel chromatography (20-25%methanol in chloroform gradient elution) to yieldO^(c)-(aminocaproyl)-ritonavir (1B) as a white solid (0.1525 g, 91%).M+H 834

EXAMPLE 10 Synthesis of O^(c)-(aminocaproyl)-saquinavir (2B)

O^(c)-(aminocaproyl)-saquinavir (2B) was prepared fromO-(N-FMOC-aminocaproyl)-saquinavir (2A) of Example 2 (0.7547 g)following the conditions described in Example 9 (0.5253 g; 89%). M+H784.3

EXAMPLE 11 Synthesis of O^(c)-(aminocaproyl)-amprenavir (3B)

O^(c)-(aminocaproyl)-amprenavir (3B) was prepared fromO-(N-FMOC-aminocaproyl)-amprenavir (3A) of Example 3 (0.2523 g)following the conditions described in Example 9 (0.1160 g; 63%). M+H619.3

EXAMPLE 12 Synthesis of O^(c)-(aminocaproyl)-indinavir (4D)

O^(c)-(N-FMOC-aminocaproyl)-O^(in),N^(in)-isopropylidinyl-indinavir (4B)synthesized as in Example 4 (0.5869 g) was stirred overnight in 50%trifluoroacetic acid in anhydrous methylene chloride (6 mL) at roomtemperature to remove the isopropylidinyl protecting group. The mixturewas evaporated to dryness under reduced pressure, the residuepartitioned between methylene chloride and saturated aqueous sodiumbicarbonate. The organic layer was separated, dried (sodium sulfate),and evaporated to a light yellow foam (0.5329 g). The foam was dissolvedin 5% piperidine in anhydrous methylene chloride (5 mL) and stirredovernight. Solvent was removed and the off-white residue purified bysilica gel chromatography (eluting with 5:1 chloroform/methanolcontaining 1% concentrated aqueous ammonium hydroxide) to giveO^(c)-(aminocaproyl)-indinavir (4D) as a colorless oil (0.2866 g; 66%overall). M+H 727.5

In another run,O^(c)-(N-FMOC-aminocaproyl)-O^(in),N^(in)-isopropylidinyl-indinavir (4B)from Example 4 (0.2301 g) was stirred 2 hours in 50% trifluoroaceticacid in anhydrous methylene chloride (3 mL) at room temperature toremove the isopropylidinyl protecting group. The mixture was evaporatedto dryness under reduced pressure and directly purified by silica gelchromatography (5% methanol in chloroform elution) to yieldO^(c)-(N-FMOC-aminocaproyl)-indinavir (4C) as a white foam (0.1603 g,70%). M+H 949.3.

EXAMPLE 13 Synthesis of O^(c)-(aminocaproyl)-nelfinavir (5C)

O^(c)-(N-FMOC-aminocaproyl)-O^(ar)-TBDMS-nelfinavir (5B) from Example 5(0.1752 g) and tetraethylammonium fluoride (0.2092 g) were stirred 2hours in anhydrous THF (10 mL) at room temperature to remove both theTBDMS and FMOC protecting groups in one step. The mixture was evaporatedto dryness under reduced pressure, redissolved in methylene chloride,washed with water then saturated aqueous sodium chloride (brine) andevaporated to dryness. The residue was purified by silica gelchromatography (elution with 2% to 10% methanol in chloroform gradientto remove front-running material, then 100% methanol to elute product)to yield O^(c)-(aminocaproyl)-nelfinavir (5C) as a white foam (0.0711 g,75%). M+H 681.3

EXAMPLE 14 O^(c)-(aminocaproyl)-lopinavir (6B)

O^(c)-(aminocaproyl)-lopinavir (6B) was prepared fromO^(c)—(N-FMOC-aminocaproyl)-lopinavir (6A, 0.100 g) of Example 6following the conditions described in Example 9, except for purificationby silica gel chromatography (10% methanol in chloroform containing 2%ammonium hydroxide) to yield product 6B (0.043 g; 56%). M+H 742.2

Another reaction, 0.300 g of (6A), performed in 10% piperidine in waterinstead of methylene chloride gave product (0.150 g; 65%) afterevaporation and silica gel chromatography as described above.

Linker Extension of O-Acylated Protease Inhibitors to Generate ActivatedHaptens EXAMPLE 15 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir (1C)

O^(c)-(aminocaproyl)-ritonavir (1B) from Example 9 (60.9 mg),triethylamine (10 μL), and succinimido-oxycarbonyl butyryl chloride(Antonian, ibid., 17.5 mg) were stirred 2 hours in anhydrous THF (6 mL)at 0° C. The mixture was evaporated to dryness under reduced pressureand directly purified by silica gel chromatography (30% THF in ethylacetate elution) to yieldO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir as awhite solid (38.8 mg, 51%). M+H 1045.2

EXAMPLE 16 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavir (1D)

First, disuccinimidyl terephthalate was prepared by the method of Kopiaet al., U.S. Pat. No. 5,667,764. To a stirring solution ofdisuccinimidyl terephthalate (21.6 mg) and triethylamine (8 μL) inanhydrous methylene chloride (8 mL) was slowly added a solution ofO^(c)-(aminocaproyl)-ritonavir (1B) from Example 9 (48.0 mg) inanhydrous methylene chloride (8 mL). The mixture was stirred 4 hours atroom temperature under argon. The mixture was evaporated to drynessunder reduced pressure and directly purified by silica gelchromatography (30% THF in ethyl acetate elution) to yieldO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavir as awhite solid (41.6 mg, 67%). M+H 1079

EXAMPLE 17 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir (2C)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir (2C) wasprepared from O^(c)-(aminocaproyl)-saquinavir (2B) of Example 10 (52.8mg) following the conditions described in Example 15, except that agradient of 5% to 10% methanol in chloroform was used as the eluent inthe silica gel chromatographic purification (48 mg; 72%). M+H 995.3

EXAMPLE 18 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavir(2F)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavir(2F) was prepared from O^(c)-(aminocaproyl)-saquinavir (2B) of Example10 (11 mg) following the conditions described in Example 16, but using2% methanol in chloroform as the eluent in the silica gelchromatographic purification (12 mg; 83%). M+H 1029.3

EXAMPLE 19 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir (3C)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir (3C) wasprepared from O^(c)-(aminocaproyl)-amprenavir (3B) of Example 11 (104.0mg)following the conditions described in Example 15, but with stirringfor 6 hours and with the use of 5% methanol in chloroform as the eluentin the silica gel chromatographic purification (80 mg; 57%). M+Na 852.4

EXAMPLE 20 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavir(3D)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavir(3D) was prepared from O^(c)-(aminocaproyl)-amprenavir (3B) of Example11 (86.5 mg) following the conditions described in Example 16, but using4% methanol in chloroform as the eluent in the silica gelchromatographic purification (70.3 mg; 58%). M+Na 886.4

EXAMPLE 21 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-indinavir (4E)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-indinavir (4E) wasprepared from O^(c)-(aminocaproyl)-indinavir (4D) of Example 12 (80.0mg) following the conditions described in Example 15, but with stirringfor 6 hours and with the use of a 5% rising to 17% methanol inchloroform gradient as the eluent in the silica gel chromatographicpurification (37.4 mg; 36%). M+H 938.6

EXAMPLE 22 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-indinavir (4F)

O^(c)-[4′-(succinimido-oxycarbonyl-benzoyl)-aminocaproyl]-indinavir (4F)was prepared from O-(aminocaproyl)-indinavir (4D) of Example 12 (90.0mg) following the conditions described in Example 16, except that 5%methanol in chloroform was used as the eluent in the silica gelchromatographic purification (61.8 mg; 51%). M+H 972.6

EXAMPLE 23 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir (5D)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir (5D) wasprepared from O^(c)-(aminocaproyl)-nelfinavir (5C) of Example 13 (60.0mg) following the conditions described in Example 15, except that a 2%rising to 5% methanol in chloroform gradient was used as the eluent inthe silica gel chromatographic purification (67.2 mg; 85%). M+H 892.5

EXAMPLE 24 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavir(5E)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavir(5E) was prepared from O-(aminocaproyl)-nelfinavir (5C) of Example 13(61.8 mg) following the conditions described in Example 16, except that5% methanol in chloroform was used as the eluent in the silica gelchromatographic purification (43.3 mg; 52%). M+H 926.6

EXAMPLE 25 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir (6C)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir (6C) wasprepared from O^(c)-(aminocaproyl)-lopinavir (6B) of Example 14 (86 mg)following the conditions described in Example 15, except forpurification by silica gel chromatography (5% methanol in chloroform)(68 mg; 62%). M+H 953.4

EXAMPLE 26 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir (6D)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir (6D)is prepared from O-(aminocaproyl)-lopinavir (6B) of Example 14 (80 mg)following the conditions described in Example 16, except forpurification by silica gel chromatography (50% tetrahydrofuran in ethylacetate) (35 mg; 33%). M+H 987.3

EXAMPLE 27 Synthesis ofO^(c)-3-[4′-(succinimido-oxycarbonyl)-phenyl-propionyl]-saquinavir (2I)

O^(c)-3-[4′-(succinimido-oxycarbonyl)-phenyl-propionyl]-saquinavir wasprepared from O^(c)-[3-(4′-carboxyphenyl)-propionyl)]-saquinavir (2H) ofExample 7 following the conditions described in Example 38 (96%). M+H944.5

EXAMPLE 28 Synthesis ofN-maleimidopropionyl-L-glutamyl-(gamma-O^(c)-saquinavir)-L-alanine (2P)

Boc-L-Glu(OBzl)OSu (Bachem), 434 mg (1 mmol) is reacted withL-Ala-O^(t)Bu.HCl, 182 mg (1 mmol) in 10 mL DMF containing triethylamine(202 mg). After stirring for 16 hours at room temperature, the reactionmixture is rotary evaporated to dryness and the residue is redissolvedin methylene chloride, washed with water, dried over sodium sulfate andevaporated to dryness. The residue is redissolved in methanol, 50 mL,and transferred to a Parr flask. 10% Pd/C catalyst (Aldrich), 50 mg, isadded and the flask is charged with 40 psi hydrogen gas on a Parrshaker. The mixture is shaken for 2 hours at room temperature or untilno further consumption of hydrogen is noted. The Parr flask is evacuatedand charged with argon gas. The mixture is filtered through Celite, andthe filtrate is rotary evaporated to give crude Boc-L-Glu-L-Ala-O^(t)Bu.

Saquinavir (335 mg), Boc-L-Glu-L-Ala-O^(t)Bu (187 mg),dicyclohexylcarbodiimide (103 mg), hydroxybenzotriazole (67.5 mg),N-ethylmorpholine (57.5 mg), and dimethylaminopyridine (61 mg) werestirred overnight in anhydrous THF (5 mL). The reaction was diluted withethyl acetate and filtered. The filtrate was washed with 2 M HCl,saturated aqueous sodium bicarbonate, and brine. The organic layer wasevaporated to dryness under reduced pressure and directly purified bysilica gel chromatography (5% methanol in methylene chloride elution) toyield N-t-butyloxycarbonyl-L-glutamyl-(gamma-O^(c)-saquinavir)-L-alaninet-butyl ester (2N) as an off-white foam (384 mg, 75%). M+H 1027

N-t-butyloxycarbonyl-L-glutamyl-(gamma-O^(c)-saquinavir)-L-alaninet-butyl ester (2N, 3.0 mg) was stirred 1 hour in 50% trifluoroaceticacid in anhydrous methylene chloride (0.05 mL) and evaporated to drynessunder reduced pressure. The residue was dissolved in anhydrous methylenechloride (0.1 mL) and stirred 30 minutes with triethylamine (1 μL) andsuccinimidyl maleimidopropionate (synthesized by the method of Ede,Tregear and Haralambidis, Bioconjugate Chem. 5, 373-378, 1994; 0.9 mg).The mixture was evaporated to dryness under reduced pressure anddirectly purified by preparative TLC (25% methanol in chloroformdevelopment) to yieldN-maleimidopropionyl-L-glutamyl-(gamma-O^(c)-saquinavir)-L-alanine (2P)as a white solid (1.7 mg, 57%). M+H 1022.3

EXAMPLE 29 Synthesis ofN-maleimidopropionyl-L-Ala-L-Glu-(gamma-O^(c)-saquinavir) (2Q)

Boc-L-Ala-L-Glu-O^(t)Bu is first synthesized using the procedure forBoc-L-Glu-L-Ala-O^(t)Bu in Example 29 substituting L-Glu(OBzl)-O^(t)Bu(Bachem) for L-Ala-O^(t)Bu and Boc-L-Ala-OSu (Bachem) forBoc-L-Glu(OBzl)-OSu, Boc-L-Ala-L-Glu(gamma-Oc-saquinavir)-O^(t)Bu (2O)was prepared from saquinavir (335 mg) and Boc-L-Ala-L-Glu-O^(t)Bu (187mg) following the conditions described in Example 28 for intermediate 2N(84%). M+H 1027

N-maleimidopropionyl-L-Ala-L-Glu-(gamma-O^(c)-saquinavir) (2Q) wasprepared from N-t-Boc-L-Ala-L-Glu-(gamma-O^(c)-saquinavir)-O^(t)Bu (2O,3.0 mg) following the conditions described in Example 28 (57%). M+H1022.3

EXAMPLE 30 Synthesis ofO^(c)-(maleimido-propionyl-aminocaproyl)-saquinavir (2M)

O^(c)-(aminocaproyl)-saquinavir (2B) from Example 10 (0.1098 g),succinimidyl maleimidopropionate (0.048 g), and triethylamine (20 μL)were stirred 45 minutes in anhydrous methylene chloride (1.5 mL). Themixture was evaporated to dryness under reduced pressure and directlypurified by silica gel chromatography (4% methanol in chloroformelution) to yield O^(c)-(maleimido-propionyl-aminocaproyl)-saquinavir(2M) as a colorless oil (0.0647 g, 49%). M+H 935.5

Alkylation of Protease Inhibitors at the Central Hydroxyl EXAMPLE 31Synthesis of O^(ar)-methoxyethoxymethyl-nelfinavir (5M)

To 28 mg (0.70 mmol) of NaH (60% in oil) was added 1 mL of hexane. Themixture was allowed to stir for 2-3 minutes under argon at roomtemperature and hexane was decanted. To the residue was added 1 mL offreshly distilled THF and 0.5 mL of anhydrous DMF followed by 50 mg(0.075 mmol) of nelfinavir mesylate as a solid in several portions. Themixture was heated at 50° C. for 45 minutes under argon and allowed tocool to room temperature. To the reaction mixture was added 12.5 μL(0.10 mmol) of 2-methoxyethoxymethyl chloride (MEM chloride) and allowedto stir at room temperature under argon for 18 hours. To the reactionmixture was added 1 mL of 50 mM potassium phosphate (pH 7.5) and themixture was concentrated under reduced pressure. To the residue wereadded 25 mL of CHCl₃ and 15 mL of 50 mM potassium phosphate (pH 7.5).The organic layer was separated and the aqueous layer was extracted withadditional 4×25 mL of CHCl₃. All the organic extracts were combined,dried (anhydrous Na₂SO₄) and concentrated. The crude product waspurified by preparative thin layer chromatography (silica gel, EMScience Cat. No. 5717-7) using 20:1 CHCl₃:MeOH as eluting solvent togive 43 mg (0.065 mmol, 88%) of O^(ar)-methoxyethoxymethyl-nelfinavir(5M) as a white solid. M+H 656.

EXAMPLE 32 Synthesis of O^(ar)-MEM-O^(c)-carboxymethyl-nelfinavir (5N)

To 14 mg (0.35 mmol) of NaH (60% in oil) was added 1 mL of hexane. Themixture was allowed to stir at room temperature under argon for 2-3minutes and hexane was decanted. To the residue 2 mL of freshlydistilled THF and 1 mL of anhydrous DMF was added. A solution of 23 mg(0.035 mmol) of 5M in 1 mL of freshly distilled THF was added to thereaction mixture. The reaction mixture was heated at 50° C. under argonfor 1 hour and allowed to cool to room temperature. To the reactionmixture was added a solution of 6.5 μL (0.043 mmol) of t-butylbromoacetate (Aldrich Chemical Co.) in 500 μL of freshly distilled THF,and the reaction mixture was allowed to stir at room temperature for 18hours under argon.

To the reaction mixture was added 1 mL of water and the mixture wasconcentrated under reduced pressure. To the residue were added 20 mL ofCHCl₃ and 15 mL of water. The organic layer was separated and theaqueous layer was extracted with additional 4×20 mL of CHCl₃. All theorganic extracts were combined, dried (Na₂SO₄) and concentrated. Thecrude product was purified by preparative thin layer chromatography(silica gel) using 20% methanol in chloroform as eluent to give 22 mg(0.031 mmol, 88%) of O^(ar)-MEM-O^(c)-carboxymethyl-nelfinavir (5N) as awhite solid. M+H 714.

EXAMPLE 33 Synthesis ofO^(ar)-MEM-O^(c)-(succinimido-oxycarbonyl-methyl)-nelfinavir (5O)

The activated ester (5O) is prepared from (5N) by following theprocedure described in Example 38.

EXAMPLE 34 Synthesis of O^(c)-(carboxymethyl)-saquinavir (2AA)

To 65 mg (1.6 mmol) of NaH (60% in oil) was added 2 mL of hexane. Themixture was allowed to stir at room temperature under argon for 2-3minutes and hexane was decanted. To the residue 2 mL of freshlydistilled THF and 1 mL of anhydrous DMF was added. Saquinavir mesylate(2, 112 mg, 0.14 mmol) was added to the reaction mixture as a solid inseveral portions. The reaction mixture was heated at 50° C. for 1 hourand allowed to cool to room temperature. To the reaction mixture asolution of 30 μL (0.203 mmol) of t-butyl bromo acetate in 500 μL offreshly distilled THF was added and the reaction was allowed to stir atroom temperature under argon for 18 hours. To the reaction mixture 1 mLof water was added and the mixture was concentrated under reducedpressure. To the residue 20 mL of water was added and the pH of thereaction was adjusted to 6 with 5% phosphoric acid. To the reactionmixture 25 mL of CHCl₃ was added. The organic layer was separated andthe aqueous layer was extracted with additional 4×25 mL of CHCl₃. Allthe organic extracts were combined, dried (Na₂SO₄) and concentrated. Theresidue was purified by flash column chromatography (silica gel) using20:1 CHCl₃:MeOH as eluent to give 68 mg (0.093 mmol, 64%) ofO^(c)-(carboxy-methyl)-saquinavir (2AA) as a white solid. M+H 729

EXAMPLE 35 Synthesis ofO^(c)-(succinimido-oxycarbonyl-methyl)-saquinavir (2BB)

The activated ester (2BB) is prepared from (2AA) by following theprocedure described in Example 38.

Derivatization of Protease Inhibitors at Positions Other than theCentral Hydroxyl EXAMPLE 36 Synthesis of ethylO^(ar)-carboxypropyl-nelfinavir (5H)

Nelfinavir (5) phenol (OH^(ar)) was selectively alkylated as follows:nelfinavir (62.5 mg) and sodium hydride (2.8 mg) were stirred 15 minutesin anhydrous DMF (1 mL) at room temperature. Ethyl 4-bromobutyrate (27.6mg, Fluka Chemical Corp.) was added and the mixture was stirred 3 hoursat room temperature. The mixture was evaporated to dryness under reducedpressure and directly purified by silica gel chromatography (3% methanolin chloroform elution) to yield ethyl O^(ar)-carboxypropyl-nelfinavir(5H) as a white solid (74.7 mg, 95%). M+H 682.4

EXAMPLE 37 Synthesis of O^(ar)-carboxypropyl-nelfinavir (5I)

Ethyl O^(ar)-carboxypropyl-nelfinavir (5H) from Example 31 (0.1440 g)and lithium hydroxide (0.0960 g) were stirred overnight in 50% aqueousTHF (10 mL). The reaction mixture was allowed to settle (two layers),the organic layer separated and evaporated to dryness under reducedpressure. A sample was purified by preparative RP-HPLC (C18; 45%acetonitrile-water containing 0.1% trifluoroacetic acid) to give theanalytical sample. The remainder was dried to yieldO^(ar)-carboxypropyl-nelfinavir (5I) as a white solid, shown by ¹H-NMRspectroscopy to be fairly clean material (0.1234 g, 89%) M+H 654.3

EXAMPLE 38 Synthesis ofO^(ar)-(succinimido-oxycarbonyl-propyl)-nelfinavir (5J)

O^(ar)-carboxypropyl-nelfinavir (5I) from Example 37 (0.1210 g, 0.185mmol), N-hydroxysuccinimide (0.0426 g, 0.37 mmol, 2 mol. equiv.; AldrichChemical Co.) and ethyl diethylaminopropyl carbodiimide hydrochloride(0.0710 g, 0.37 mmol, 2 mol. equiv.; Sigma Chemical Co) was stirred 2hours in 10% anhydrous DMF-methylene chloride (9 mL). The mixture wasevaporated to dryness under reduced pressure and purified by silica gelchromatography (3% methanol in chloroform elution) followed bypreparative RP-HPLC (C18; 45% acetonitrile-water containing 0.1%trifluoroacetic acid) to yieldO^(ar)-(succinimido-oxycarbonyl-propoxy)-nelfinavir (5J, 0.0681 g, 49%).M+H 751.3

Another reaction performed as above but using 5I (0.2764 g) followed bysilica gel chromatography (3% methanol in chloroform elution) gave crudebut fairly clean product (5J, 0.3526 g) as an oil.

EXAMPLE 39 Synthesis ofO^(ar)-(succinimido-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-propyl)-nelfinavir(5K)

O^(ar)-(succinimido-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-propyl)-nelfinavir(5K) was prepared fromO^(ar)-(succinimido-oxycarbonyl-propoxy)-nelfinavir (5J, 0.32 g) ofExample 38 following the conditions of Example 41 (0.0657 g; 32%). M+H950.4

EXAMPLE 40 Synthesis of N-(succinimido-oxycarbonyl-butyryl)-amprenavir(3G)

Amprenavir (3, 0.1517 g) and succinimido-oxycarbonyl butyryl chloride(0.0817 g) were stirred overnight in anhydrous DMF (3 mL) at 50° C. Themixture was evaporated to dryness under reduced pressure and directlypurified by silica gel chromatography (15% THF in ethyl acetate elution)to yield N-(succinimido-oxycarbonyl-butyryl)-amprenavir (3G) as a whitesolid (0.1395 g, 61%). M+Na 739.2. Spectral data (¹H-NMR) was compatiblewith functionalization at the aniline nitrogen.

EXAMPLE 41 Synthesis ofN-(succinimidyl-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-glutaryl)-amprenavir(3H)

(a) N-(succinimido-oxycarbonyl-propionyl)-amprenavir (3G) from Example40 (131.5 mg) and glycyl-glycyl-4-aminobutyric acid (43.4 mg, BachemCalifornia Inc., CA) were stirred 7 hours in 25% aqueous borate (pH 10)in THF (5 mL). The mixture was evaporated to dryness under reducedpressure and directly purified by preparative RP-HPLC (C18; 45%acetonitrile-water containing 0.1% trifluoroacetic acid) to yieldN-(3-carboxypropylamino-^(co)glycyl-glycyl-glutaryl)-amprenavir as awhite solid (98.2 mg, 65%). M−H 817.4

(b) N-(4-carboxypropylamino-^(co)glycyl-glycyl-glutaryl)-amprenavir(40.9 mg), N-hydroxysuccinimide (11.5 mg), and ethyl dimethylaminopropylcarbodiimide (19.2 mg) were stirred 5 hours in 20% anhydrous DMF inmethylene chloride (2.5 mL). The mixture was evaporated to dryness underreduced pressure and directly purified by silica gel chromatography (12%methanol in chloroform elution) to yieldN-(succinimidyl-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-glutaryl)-amprenavir(3H) as a white foam (37.9 mg, 83%). M+H 938.4

Urethane Derivatization of Protease Inhibitors EXAMPLE 42 Synthesis ofethyl O^(c)-(carboxymethylaminocarbonyl)-saquinavir (2J)

Saquinavir methanesulfonate (2, 76.7 mg), ethyl isocyanatoacetate (23.0mg, Aldrich Chemical Co.), and triethylamine (30 μL) were stirred 5 daysin anhydrous DMF (1 mL) at 50° C. The mixture was evaporated to drynessunder reduced pressure and directly purified by silica gelchromatography (5% methanol in chloroform elution) to yield ethylO^(c)-(carboxymethylaminocarbonyl)-saquinavir (2J) as a white solid(32.3 mg, 40%). M+H 800.4

EXAMPLE 43 Synthesis of O^(c)-(carboxymethylaminocarbonyl)-saquinavir(2K)

Ethyl O^(c)-(carboxymethylaminocarbonyl)-saquinavir (2J) from Example 36(0.1600 g) and lithium hydroxide (0.0960 g) were stirred 1 hour 50%aqueous THF (10 mL). The organic layer was isolated, dried withanhydrous sodium sulfate, and evaporated to dryness under reducedpressure to yield O^(c)-(carboxymethylaminocarbonyl)-saquinavir (2K) asa white foam (0.1403 g, 91%). M+H 772.3.

EXAMPLE 44 Synthesis ofO^(c)-(succinimido-oxycarbonyl-methylaminocarbonyl)-saquinavir (2L)

O^(c)-(carboxymethylaminocarbonyl)-saquinavir (2K) of Example 43 (0.1930g), succinimidyl tetramethyluronium tetrafluoroborate (0.1882 g, AldrichChemical Co.) and diisopropylethylamine (0.15 mL) were stirred overnightin anhydrous THF (10 mL). HPLC-MS showed 80% complete reaction, productpeak (2L) M+H 869.3.

EXAMPLE 45 Synthesis ofO^(c)-[(4-methoxycarbonylphenyl)-methylamino-^(co)-glycyl-carbonyl]-saquinavir(2W)

O^(c)-(carboxymethylaminocarbonyl)-saquinavir (2K) from Example 43(0.1929 g) and succinimidyl tetramethyluronium tetrafluoroborate (0.1505g) were stirred overnight in anhydrous tetrahydrofuran (10 mL)containing diisopropylethylamine (0.15 mL) to give (2L) in situ.Methyl-4-aminomethylbenzoate hydrochloride (0.1008 g, Aldrich ChemicalCo.) and diisopropylethylamine (0.15 mL) were added and stirred 3 hours.The mixture was evaporated to dryness under reduced pressure anddirectly purified by silica gel preparative TLC (50% ethyl acetate and2% methanol in chloroform) to yield 2W as a white solid (0.1905 g, 83%).M+H 919.4

EXAMPLE 46 Synthesis ofO^(c)-[(4-carboxyphenyl)-methylamino-^(co)-glycyl-carbonyl]-saquinavir(2X)

O^(c)-[(4-methoxycarbonylphenyl)-methylamino-^(co)-glycyl-carbonyl]-saquinavir(2W) from Example 45 (0.232 g) was dissolved in methanol (10 mL).Lithium hydroxide (0.154 g) and water (2.5 mL) were added and thereaction was stirred overnight. The reaction mixture was extracted withmethylene chloride, and the organic layer was dried with anhydroussodium sulfate and evaporated to dryness under reduced pressure. Theresidue was purified by silica gel chromatography (10% methanol inchloroform containing 2% acetic acid) to yield 2X as a white solid(0.100 g, 44%). M+H 772.3

EXAMPLE 47 Synthesis ofO^(c)-[4-(succinimido-oxycarbonyl-phenyl)-methylamino-^(co)-glycyl-carbonyl]-saquinavir(2Y)

O^(c)-[(4-succinimido-oxycarbonyl-phenyl)-methylamino-^(co)-glycyl-carbonyl]-saquinavir(2Y) was prepared fromO^(c)-[(4-carboxyphenyl)-methylamino-^(co)-glycyl-carbonyl]-saquinavir(2X) from Example 46 (85 mg) following the conditions described inExample 38. M+H 1002.3

EXAMPLE 48 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-phenyl-aminocarbonyl]-saquinavir(2U)

50 mg (65.2 μmol) of saquinavir mesylate (2) in 5 mL freshly distilledDMF and 9 μL (65.2 μmol) triethylamine were stirred for about 10 minutesat ambient temperature until a clear solution was obtained. 236.1 mg(1.3 mmol) 4-isocyanatobenzoyl chloride were added and the mixtureturned red instantly. After standing at room temperature for 2 hours a 1μL sample of the solution was injected into analytical HPLC (Vydac C18column, 300 Å, 5 μm, 4.6×250 mm; eluent A: Millipore water/0.1%trifluoroacetic acid, eluent B: acetonitrile/0.1% trifluoroacetic acid;gradient of 0% B in A, rising to 60% B in A, over 60 minutes). Thechromatography profile at 226 nm showed nearly complete derivatizationof the educt (t_(s)=45.1 minutes) and formation of urethane (t_(s)=48.3min.) together with some by-products.

22 mg of crude product was isolated from the mixture by preparative HPLC(Vydac C18 column, 300 Å, 15-20 μm, 50×250 mm; eluent A: Milliporewater/0.1% trifluoroacetic acid, eluent B: 80% acetonitrile/0.1%trifluoroacetic acid; gradient of 0% B in A, rising to 70% B in A, over140 minutes). The appropriate fractions eluting at about 62-65% B werepooled, lyophilized and subjected to a second chromatography step(modified gradient: gradient of 0% B in A, rising to 75% B in A, over120 minutes). 10 mg (18%) of slightly red pure product were obtainedfrom fractions 16 and 17. MALDI-TOF MS of purified carboxylic acidintermediate 2T. M+H 834, M+Na 856

10 mg (12 μmol) of O^(c)-(4-carboxyphenylaminocarbonyl)-saquinavir (2T)was dissolved in 500 μL freshly distilled DMF and 1.7 mg (15 μmol)N-hydroxysuccinimide (NHS) and 2.9 mg (15 μmol)ethyl-dimethylaminopropyl carbodiimide (EDC) were added. The solutionwas stirred 5 hours at room temperature under argon, then again 1.7 mg(15 μmol) NHS and 2.9 mg EDC were added. The mixture was stirred furtherand allowed to react 2.5 days at room temperature. HPLC showed formationof NHS ester 2U, which was not isolated but used in situ for furtherreactions.

EXAMPLE 49 Synthesis of ethylO^(c)-(carboxymethylaminocarbonyl)-O^(ar)-TBDMS-nelfinavir (5P)

O^(ar)-TBDMS-Nelfinavir (5A) of Example 5 (0.102 g), ethylisocyanatoacetate (42 μL), and triethylamine (55 μL) were stirred 3.5days in anhydrous DMF (2 mL) at 50° C. The mixture was evaporated todryness under reduced pressure and purified first by silica gelchromatography (2% methanol in chloroform) followed by preparativeRP-HPLC (C18) (60% acetonitrile-water containing 0.1% trifluoroaceticacid/30 minutes rising to 70% acetonitrile-water containing 0.1%trifluoroacetic acid over 30 minutes) to give recovered startingmaterial 5A (0.0503 g; 43%) followed by the product 5P (0.0623 g; 45%)after lyophilization of the appropriate fractions. M+H 811.4

EXAMPLE 50 Synthesis of O^(c)-(carboxymethylaminocarbonyl)-nelfinavir(5Q)

Ethyl O^(c)-(carboxymethylaminocarbonyl)-O^(ar)-TBDMS-nelfinavir (5P) ofExample 49 (56.5 mg) in 3.5 mL of 1:1 tetrahydrofuran-water was treatedwith 50 mg of lithium hydroxide monohydrate and the reaction stirred for4 hours. The layers were allowed to settle, the organic layer isolated,dried with sodium sulfate and evaporated. The residue was largelyredissolved in acetonitrile (5 mL), filtered and purified by preparativeRP-HPLC (C18) (35% acetonitrile in water containing 0.1% trifluoroaceticacid) to give the O^(ar)-deprotected product 5Q (24.3 mg; 52%) M+H 669.2

EXAMPLE 51 Synthesis ofO^(c)-[(3-carboxypropyl)amino-^(co)-glycyl-glycyl-glycyl-carbonyl)-nelfinavir(5R)

O^(c)-(carboxymethylaminocarbonyl)-nelfinavir (5Q) of Example 50 (20.4mg), succinimidyl tetramethyluronium tetrafluoroborate (12.0 mg), anddiisopropylethylamine (8 μL) were stirred overnight in anhydrous THF(1.5 mL) for 5.5 hours. LC/MS showed the presence of the correspondingNHS ester together with some starting material.Glycyl-glycyl-4-aminobutyric acid (7.0 mg) was added followed by 50 mMphosphate buffer (pH 10) until a clear solution was obtained. Afterstirring overnight the reaction was concentrated to ˜1 mL, the milkyresidue diluted with acetonitrile and sonicated to give a clear solutionwhich was purified by preparative RP-HPLC (C18) (30% acetonitrile inwater for 30 minutes, 30% to 45% acetonitrile in water over 30 minutes,45% to 90% acetonitrile in water over 30 minutes, all containing 0.1%trifluoroacetic acid) to give the product 5R from the main peak afterlyophilization. (12.6 mg, 48%) M+H 868.4

EXAMPLE 52 Synthesis ofO^(c)-(succinimido-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-glycyl-carbonyl)-nelfinavir(5S)

O^(c)-(Succinimido-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-glycyl-carbonyl)-nelfinavir(5S) is synthesized fromO^(c)-[(3-carboxypropyl)amino-^(co)-glycyl-glycyl-glycyl-carbonyl)-nelfinavir(5R) of Example 51 following the conditions of Example 41(b). M+H 964.4

Conjugation of Protease Inhibitors to Small Molecular Weight LabelsEXAMPLE 53 Synthesis ofO^(c)-(fluoresceinyl-glycinamidyl-butyryl-aminocaproyl)-saquinavir (2V)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir (2C)from Example 17 (10.0 mg) and fluoresceinyl glycinimide (5.0 mg,Molecular Probes, OR) is stirred overnight in 3% triethylamine-pyridine(0.1 mL). The mixture was evaporated to dryness under reduced pressureand directly purified by preparative RP-HPLC (C18; 50%acetonitrile-water containing 0.1% trifluoroacetic acid) to yieldO^(c)-(fluoresceinyl-glycinamidyl-butyryl-aminocaproyl)-saquinavir (2V;7.6 mg, 75%). M+H 1284.6

EXAMPLE 54 Synthesis ofO^(c)-(fluoresceinyl-glycinamidyl-butyryl)-ritonavir (1I)

O^(c)-(fluoresceinyl-glycinamidyl-butyryl)-ritonavir (1I) was preparedfrom O^(c)-(succinimido-oxycarbonyl-butyryl)-ritonavir (1G) of Example 8following the conditions described in Example 53 (8.4 mg; 69%). M+H1221.4

EXAMPLE 55 Synthesis ofO^(c)-[4′-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminocaproyl]-indinavir(4I)

5.0 mg ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl-indinavir (4F)from Example 22 were dissolved in 5.0 mL freshly distilled DMF. 13.6 mgof 1-biotinylamino-3,6-dioxa-octaneamine (biotin-DADOO, Roche AppliedScience, Cat. No. 1112074-103) and 5.6 μL triethylamine were added, andthe resulting clear solution was stirred under argon overnight. HPLCcontrol showed complete reaction after 20 hours. DMF was removed on arotavapor (high vacuum, much less than 1 Torr pressure, water bath 30°C.). The remaining oily product was dissolved in 0.5 mL DMSO, filteredand injected into a preparative HPLC system (Vydac C18 column, 300 Å,15-20 μm, 50×250 mm; eluent A: Millipore water/0.1% trifluoroaceticacid, eluent B: 80% acetonitrile/0.1% trifluoroacetic acid; gradient of0% B in A rising to 70% B in A over 140 minutes), the appropriatefractions containing pure product were pooled and lyophilized. Structurewas confirmed by MALDI-TOF-MS (M=1231). Yield: 3.5 mg (2.84 μmol, 55% oftheoretical yield)

EXAMPLE 56 Synthesis ofO^(c)-[4′-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminocaproyl]-amprenavir(3J)

An amprenavir-biotin conjugate (3J,O^(c)-[4′-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminocaproyl]-amprenavir)was synthesized using the activated haptenO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl-amprenavir (3D)from Example 20 in the procedure described in Example 55 above.Structure was confirmed by MALDI-TOF-MS (M=1123). Yield: 1.8 mg (1.60μmol, 31% of theoretical yield)

EXAMPLE 57 Synthesis ofO^(c)-[4′-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminocaproyl]-lopinavir(6G)

A lopinavir-biotin conjugate (6G,O^(c)-[4′-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminocaproyl]-lopinavir)was synthesized using the activated haptenO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir (6D)from Example 26 in the procedure described in Example 55 above.Structure was confirmed by MALDI-TOF-MS (M=1246). Yield: 0.6 mg (0.48μmol, 10% of theoretical yield)

EXAMPLE 58 Synthesis ofO^(c)-[4′-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminocaproyl]-ritonavir(1J)

A ritonavir-biotin conjugate (1J,O^(c)-[4′-(1-biotinyl-amino-3,6-dioxa-octylamino)-terephthaloyl-aminocaproyl]-ritonavir)was synthesized using the activated haptenO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl-ritonavir (1D)from Example 22 in the procedure described in Example 55 above.Structure was confirmed by MALDI-TOF-MS (M=1338). Yield: 5.4 mg (4.03μmol, 87% of theoretical yield)

Conjugation of Protease Inhibitors to Proteins EXAMPLE 59 Synthesis ofConjugate 2S ofN-maleimidopropionyl-L-alanyl-L-(gamma-O^(c)-saquinavir)-glutamic Acidwith 2 IT Modified Bovine Serum Albumin

Bovine serum albumin (30 mg) and 2-iminothiolane (2-IT) hydrochloride(0.5 mg, Pierce Biotechnology Inc., IL) were allowed to stand 1 hour inthe dark in 10 mM potassium phosphate, 0.1 M sodium chloride, 1 mM EDTA,pH 8.0 (3 mL). The mixture was desalted by gel filtration on a PD-10column (Amersham-Pharmacia, NJ) eluting with 10 mM potassium phosphate,0.1 M sodium chloride, 1 mM EDTA, pH 8.0. The appropriate fractions werecollected, adjusted to pH 7.2, andN-maleimidopropionyl-L-alanyl-L-(gamma-O^(c)-saquinavir)-glutamic acid(2Q) from Example 29 (1 mg) dissolved in methanol (0.2 mL) was added.The mixture was allowed to stand 2 hours in the dark, quenched withethyl maleimide (0.5 mg, Sigma Chemical Co.), and desalted by gelfiltration on a PD-10 column (10 mM potassium phosphate, 0.1 M sodiumchloride, 1 mM EDTA, pH 8.0, elution). Protein quantification byCoomassie Blue protein assay (Bio-Rad Laboratories, CA; modifiedBradford protein assay) showed quantitative recovery of protein at 4.3mg/mL. UV difference spectroscopy showed the ratio of hapten to BSA tobe 1:1.

EXAMPLE 60 Synthesis of Conjugate 2R ofN-maleimidopropionyl-L-glutamyl-(gamma-O^(c)-saquinavir)-L-alanine withSATP-Modified KLH

Keyhole limpet hemocyanin (CALBIOCHEM, CN Biosciences, San Diego,Calif.; slurry in 65% ammonium sulfate) was dialyzed exhaustivelyagainst 50 mM potassium phosphate buffer pH 7.5 (>8 buffer changes;dilution factor more than 10¹⁰) at room temperature (2-3 buffer changes)then at 4° C. The retentate was lyophilized almost to dryness, thenreconstituted with an appropriate volume of 50 mM phosphate to givepurified KLH at a relatively high concentration. Unused portions of thepurified KLH were frozen and stored at −20° C. until needed.

Purified keyhole limpet hemocyanin (20 mg) and N-succinimidylS-acetylthiopropionate (SATP, 10 mg, Pierce Biotechnology, Inc.) wereallowed to stand 1 hour in 50 mM potassium phosphate, 1 mM EDTA, pH 7.5,and desalted by gel filtration on a PD-10 column (Amersham-Pharmacia)eluting with 50 mM potassium phosphate, 1 mM EDTA, pH 7.5. Derivatizedprotein (10 mg) was allowed to stand 2 hours in the dark in 50 mMpotassium phosphate, 2.5 mM EDTA, 50 mM hydroxylamine hydrochloride, pH7.5, and desalted by gel filtration (50 mM potassium phosphate, 5 mMEDTA, pH 7.2 elution).N-maleimidopropionyl-L-glutamyl-(gamma-O^(c)-saquinavir)-L-alanine (2P)from Example 28 (6 mg) dissolved in DMSO (1 mL) was added and thereaction was stirred 16 hours. Ethyl maleimide (0.5 mg) was added andthe reaction was stirred 8 hours. The mixture was sequentially dialyzedagainst 30%, 20%, 10% and 0% DMSO in 50 mM potassium phosphate, pH 7.5at room temperature, followed by dialysis against 50 mM potassiumphosphate, pH 7.5 at 4° C. Protein quantification by Coomassie Blueshowed quantitative recovery of protein at 1.6 mg/mL. UV differencespectroscopy showed up to 25% lysine substitution by hapten.

EXAMPLE 61 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavirConjugate with BSA (2D)

Bovine serum albumin (30 mg) andO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir (2C)from Example 17 (1 mg) were stirred 2 days in 30% DMSO in 50 mMpotassium phosphate, pH 7.5 (1.5 mL), at room temperature. The mixturewas sequentially dialyzed against 30%, 20%, 10% and 0% DMSO in 1 liter50 mM potassium phosphate, pH 7.5, at room temperature, followed bydialysis against 1 liter 50 mM potassium phosphate, pH 7.5, at 4° C.Protein quantification by Coomassie Blue showed quantitative recovery ofprotein at 10.4 mg/mL. UV difference spectroscopy showed the ratio ofhapten to BSA to be 1:1.

EXAMPLE 62 Synthesis ofO^(c)-[(4′-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavirConjugate with BSA (2G)

O^(c)-[(4′-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavir BSAconjugate was prepared from bovine serum albumin (30 mg) andO^(c)-[(4′-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-saquinavir(2F) from Example 18 (1 mg) following the conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 10.4 mg/mL. UV difference spectroscopy showed theratio of hapten to BSA to be 1:1.

EXAMPLE 63 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavirConjugate with KLH (2E)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir KLHconjugates was prepared from purified keyhole limpet hemocyanin (30 mg)and O^(c-)(succinimido-oxycarbonyl-butyryl-aminocaproyl)-saquinavir (2C)from Example 17 (10 mg) following the general conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 10.9 mg/mL. Amine quantification bytrinitrobenzenesulfonic acid (TNBS, Sigma Chemical Co.) colorimetricassay showed 60% lysine modification.

EXAMPLE 64 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir Conjugatewith LPH (1E)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir LPHconjugate was prepared from horseshoe crab hemocyanin (LPH, 30 mg; SigmaChemical Co.) andO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir (1C) fromExample 15 (7 mg) following the general conditions described in Example61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 7.9 mg/mL. Amine quantification by TNBScolorimetric assay showed 26% lysine modification.

EXAMPLE 65 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavirConjugate with BSA (1F)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavir BSAconjugate was prepared from bovine serum albumin (30 mg) andO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-ritonavir (1D)from Example 16 (1 mg) following the general conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 10.3 mg/mL. TNBS colorimetric assay showed theratio of hapten to BSA to be 2:1.

EXAMPLE 66 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl)-ritonavir Conjugate with KLH(1H)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir KLHconjugate was prepared from purified keyhole limpet hemocyanin (30 mg)and O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir (1G)from Example 8 (10 mg) following the general conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 11.9 mg/mL. Amine quantification by TNBScolorimetric assay showed 60% lysine modification.

EXAMPLE 67 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavirConjugate with BSA (3E)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir KLHconjugate was prepared from purified keyhole limpet hemocyanin (30 mg)and O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavir (3C)from Example 19 (8 mg) following the general conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 6.8 mg/mL. Amine quantification by TNBScolorimetric assay showed 20% lysine modification.

EXAMPLE 68 Synthesis ofO^(c)-[(succinimido-oxycarbonyl)-butyryl-aminocaproyl]-indinavirConjugate with KLH (4G)

O^(c)-[(4′-succinimido-oxycarbonyl-butyryl)-aminocaproyl]-indinavir KLHconjugate was prepared from purified keyhole limpet hemocyanin (30 mg)and O^(c)-[(succinimido-oxycarbonyl-butyryl)-aminocaproyl]-indinavir(4E) from Example 21 (9 mg) following the general conditions describedin Example 61. Protein quantification by Coomassie Blue showedquantitative recovery of protein at 7.4 mg/mL. Amine quantification byTNBS colorimetric assay showed 20% lysine modification.

EXAMPLE 69 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavirConjugate with BSA (3F)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavir BSAconjugate was prepared from bovine serum albumin (30 mg) andO^(c)-[(4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-amprenavir(3D) from Example 20 (1 mg) following the general conditions describedin Example 61. Protein quantification by Coomassie Blue showedquantitative recovery of protein at 11.5 mg/mL. UV differencespectroscopy showed the ratio of hapten to BSA to be 2:1.

EXAMPLE 70 Synthesis ofO^(c)-[(4′-succinimido-oxycarbonyl-benzoyl)-aminocaproyl]-indinavirConjugate with BSA (4H)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-indinavir BSAconjugate was prepared from bovine serum albumin (30 mg) andO^(c)-[4′-(succinimido-oxycarbonyl-benzoyl)-aminocaproyl]-indinavir (4F)from Example 22 (1 mg) following the general conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 10.8 mg/mL. UV difference spectroscopy showed theratio of hapten to BSA to be 2:1.

EXAMPLE 71 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavirConjugate with KLH (5F)

O^(c)-[(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir KLHconjugate was prepared from purified keyhole limpet hemocyanin (30 mg)and O^(c)-[(succinimido-oxycarbonyl-butyryl-aminocaproyl)-nelfinavir(5D) from Example 23 (9 mg) following the general conditions describedin Example 61. Protein quantification by Coomassie Blue showedquantitative recovery of protein at 9.7 mg/mL. Amine quantification byTNBS colorimetric assay showed 36% lysine modification.

EXAMPLE 72 Synthesis ofO^(c)-(4′-[succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavirConjugate with BSA (5G)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavir BSAconjugate was prepared from bovine serum albumin (30 mg) andO^(c)-[(4′-succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-nelfinavir(5E) from Example 24 (1 mg) following the general conditions describedin Example 61. Protein quantification by Coomassie Blue showedquantitative recovery of protein at 10.9 mg/mL. UV differencespectroscopy showed the ratio of hapten to BSA to be 2:1.

EXAMPLE 73 Synthesis ofO^(ar)-(succinimido-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-propoxy)-nelfinavirConjugate with KLH (5L)

O^(ar)-(succinimido-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-propoxy)-nelfinavirKLH conjugate was prepared from keyhole limpet hemocyanin (30 mg) andO^(ar)-(succinimido-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-propoxy)-nelfinavir(5K, 10 mg) of Example 39 following the general conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 14.6 mg/mL. Amine quantification by TNBScolorimetric assay showed 57% lysine modification.

EXAMPLE 74 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir Conjugatewith KLH (6F)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir KLHconjugate was prepared from keyhole limpet hemocyanin (40 mg) andO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir (6C) fromExample 25 (16 mg) in 40% dimethyl sulfoxide in 50 mM potassiumphosphate, pH 7.5 (3.4 mL), in a similar manner to Example 61, followedby sequential dialysis against 40%, 30%, 20%, 10% and 0% DMSO in 50 mMpotassium phosphate, pH 7.5 at room temperature, followed by dialysisagainst 50 mM potassium phosphate, pH 7.5 at 4° C. Proteinquantification by Coomassie Blue showed quantitative recovery of proteinat 6.9 mg/mL. Amine quantification showed 38% lysine modification.

EXAMPLE 75 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavirConjugate with BSA (6E)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir BSAconjugate was prepared from bovine serum albumin (93 mg) andO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-lopinavir (6D)from Example 26 (3 mg) following the general conditions described inExample 61. Protein quantification by Coomassie Blue showed quantitativerecovery of protein at 11.1 mg/mL. UV difference spectroscopy showed theratio of hapten to BSA to be 2:1.

EXAMPLE 76 Synthesis ofN-(succinimidyl-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-glutaryl)-amprenavirConjugate with KLH (3I)

N-(succinimidyl-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-glutaryl)-amprenavirKLH conjugate was prepared from purified keyhole limpet hemocyanin (30mg) andN-(succinimidyl-oxycarbonyl-propylamino-^(co)-glycyl-glycyl-glutaryl)-amprenavir(3H) from Example 41 (9 mg) following the general conditions describedin Example 61. Protein quantification by Coomassie Blue showedquantitative recovery of protein at 8.7 mg/mL. Amine quantification byTNBS colorimetric assay showed 40% lysine modification.

Development of Antibodies to Protease Inhibitors EXAMPLE 77 AntibodyResponse to Saquinavir KLH Immunogen

Saquinavir-KLH (2E) was used to immunize mice of both the C57 Black andSwiss Webster strains. The doses and routes of immunization were thesame for both strains of mice. The immunization schedule is given inTable 1.

TABLE 1 Immunization Schedule Date Dose/Route Adjuvant Day 0  75 μg FP &IP Complete Freund's Day 25 100 μg FP & IP Incomplete Freund's Day 53100 μg IP Incomplete Freund's FP = Foot pad; IP = Intraperitoneal

Blood samples were taken from each mouse by retro-orbital bleedsthirteen days after the last immunization. The blood was immediatelycentrifuged and the serum drawn off and stored in a micro-vial afterbeing diluted 10 times with phosphate buffered saline with 0.02%thimerosal preservative.

The next day an ELISA was conducted to establish the titers of antibodypresent. The ELISA consisted of microtiter plates coated with differentsaquinavir-BSA conjugates, all at 1 μg/mL in bicarbonate buffer (0.1 M,pH 9.6, 100 μL/well, 4° C. overnight). After coating the plates wereemptied and 200 μL of post coat solution consisting of Tris buffer, 1%gelatin hydrolysate, 2% sucrose and 0.17% TWEEN-20 emulsifying agent(ICI Americas, Inc.) was added. This was incubated at 37° C. for 1 hourto block any uncoated regions of the wells. The sera were tested bycarrying out a pre-dilution to 1000, then serial dilutions down eachcolumn at a 1:3 ratio. The volume of diluted serum in each well was 100μl, which was allowed to incubate at 37° C. in a humidified containerfor 1 hour and 20 minutes. The plates were then washed with phosphatebuffered saline, and 100 μL of goat anti-mouse IgG-HRP (horseradishperoxidase) conjugate (Zymed, Inc., diluted 1:5000 in PBS) was added toeach well. The plates were again incubated for 2 hours under the sameconditions, then washed again. Development consisted of addition of 100μL of K-BLUE SUBSTRATE (Neogen Corporation) to each well, and incubationat room temperature in the dark for 30 minutes. Development was stoppedby the addition of 100 μL of 1N HCl to each well. The optical densitiesof each plate were read with a microplate reader at 450 nm and capturedto a computer.

The serum titers when examined with respect to saquinavir-BSA conjugate2D with the same linker structure and position as the immunogen weresubstantially higher than for the other conjugates, indicating there wassome linker recognition in the polyclonal antibody population. Asillustrated in FIG. 15, titers decreased as the-structure and positionof the linker differed from the immunogen. FIG. 15 is a graph of titersof mouse #333 serum using saquinavir conjugates 2G, 2W, 2D and 2S.(Note: Preparation of conjugate 2W is described in previously citedcopending application EP 1 207 394 A2 as Example V.) The optical densityat 450 nm read at 30 minutes is plotted on the Y-axis and serumdilutions are plotted on the X-axis.

From these analyses, it was clear that the saquinavir-KLH conjugate wassuitable for use in raising polyclonal antibodies and could alsotherefore be used for the development of monoclonal antibodies.

EXAMPLE 78 Development of Monoclonal Antibodies to Saquinavir

Female Swiss-Webster mice, at least 3 months of age, were used forimmunizations. The KLH immunogen 2E was emulsified in 50% CompleteFreund's Adjuvant, 50% saline, at a final concentration of 0.75 mg/ml.Each mouse was injected twice with 10 μL subcutaneously in the rearthigh region, and with 90 μl in the peritoneal space. Twenty five dayslatter, similar injections were given in the same routes, using Freund'sIncomplete Adjuvant and a concentration of 1 mg/ml, total volume permouse was 0.1 ml. Thirteen days later each mouse was bledretro-orbitally to obtain a serum sample for analysis. A thirdimmunization was administered 49 days later, identical to the secondformulation. The mouse selected for use in fusion was given a boosterimmunization thirteen days later, identical to that of the second andthird injections. Four days later, the mouse was used for cell fusion todevelop monoclonal antibody secreting hybridomas.

The conjugate 2D featuring the linker homologous to the immunogen showedthe greatest efficacy of binding of serum antibodies. The binding wasdirectly related to the degree of homology of the conjugate linker tothat of the immunogen. Efficacy of binding was found to be, from thestrongest to the weakest, 2D>2G>2S>2W. Based on the observations made inanalyzing the serum antibodies above, it was decided to devise astrategy for screening of monoclonal antibodies in the fusion phase ofthe work in which the effect of linker homology could be distinguishedand only those clones showing little or no linker preference would beselected.

The strategy featured two tactics. First, antibody binding would betested using a linker shown by the above analysis to provide less thanmaximal binding of the sera antibodies. Second, a second well coatedwith the same conjugate, in which 400 ng/mL of free drug was included,would be employed to estimate the competitive effect of the drug onbinding. The result would allow the selection of only those monoclonalswhich competitively bound the free drug (i.e., without any linkerattached).

The mouse selected for fusion was killed via exsanguination. Thepopliteal, inguinal, subclavial and deep inguinal lymph nodes and spleenwere harvested and pooled. The tissues were ground between two sterileglass slides to release the lymphocytes. One-half of the resultinglymphocyte suspension was used to fuse with the F0 myeloma cell line(ATCC CRL 1646), the remaining half was fused with the P3 myeloma (bothmyelomas were from ATCC).

Fusion consisted with adding myeloma cells (⅕ the number of lymphocytes)to the lymphocytes, washing via centrifugation, resuspension inserum-free warm Iscove's modified Dulbecco's media (IMDM), andre-centrifugation. The centrifuge tubes containing the resulting pelletswere gently tapped to loosen the cells, then 1 mL of warmed PEG/DMSOsolution (Sigma Chemicals) was slowly added with gentle mixing. Thecells were kept warm for 1.5 minutes, after which pre-warmed serum-freeIMDM was added at the following rates: 1 ml/min, 2 ml/min, 4 ml/min, 10ml/min, then the tube was filled to 50 ml, sealed and incubated for 15minutes. The cell suspensions were centrifuged, the supernatantdecanted, and IMDM containing 10% fetal calf serum was added. The cellswere centrifuged once again, and resuspended in complete cloning medium.This consisted of IMDM, 10% FCS, 10% Condimed H1 (Roche MolecularSystems), 4 mM Glutamine, 50 μM 2-mercaptoethanol, 40 μM ethanolamine,pen/strep antibiotics. The cells were suspended at a density of 4×10⁵lymphocytes/ml, distributed 100 μL/well into sterile 96-well sterilemicroculture plates and incubated at 37° C. in 5% CO₂ for 24 hours. Thenext day, 100 μL of hypoxanthine-methotrexate-thymidine (HMT) selectivemedium (cloning medium+1:25 HMT supplement from Sigma Chemicals) wasadded. On the 6^(th) day of incubation, approximately 150 μL of mediawas drawn from each well using a sterile 8-place manifold connected to alight vacuum source. One hundred fifty microliters ofhypoxanthine-thymidine (HT) media was then added. This consists ofcloning medium+1:50 HT supplement (Sigma Chemicals). The plates werereturned to the incubator and inspected daily for signs of growth. Whengrowth was judged sufficient, wells were screened for antibodyproduction via ELISA.

Microplates were coated with 100 μL saquinavir-BSA conjugate at 1 μg/mLin 0.1 M carbonate buffer, pH 9.5 for 1 hour at 37° C. (humidified). Theplates were then emptied and filled with a post-coat solution. Theplates were incubated for an additional 1 hour at 37° C. (humidified)after which they were washed with phosphate-buffered saline containing0.1% TWEEN 20. The plates were then filled with a 2% sucrose solution in0.15 M Tris, pH 7.2-7.4 briefly, then emptied and allowed to air dry atroom temperature. When dried, the plates were packed in zip-lock bagscontaining several desiccant pillows, sealed and stored at 4° C. untiluse.

When the growing clones were judged ready for testing, 25 μL ofsupernatant from the wells were taken and transferred to 96-wellflexible plates. Culture medium was added to each well to provide a 1:10dilution of the media sample. Two saquinavir-BSA coated wells were usedfor each culture well tested. One well received 50 μL of PBS buffer, theother received 50 μL of PBS containing saquinavir drug at aconcentration of 800 ng/ml. Fifty microliters of the diluted sample weretransferred to each of two of the coated wells above. The plates wereincubated covered for 1 hour at 37° C., then washed with PBS-TWEEN. Thewells were then filled with 100 μL of goat anti-mouse IgG-HRP conjugate(Zymed Labs) diluted 1:5,000 in PBS-TWEEN and the plates re-incubatedfor 1 hour. The plates were then washed again, and 100 μL of K-BLUESUBSTRATE (Neogen Corp) were added to each well. This was allowed todevelop for 5-15 minutes, the reaction being stopped by the addition of100 μL of 1 N HCl. Color was read via a microplate reader at 450 nm andcollected by computer for analysis. Criteria for selection were bindingto the saquinavir-BSA conjugate and significant inhibition of binding inthe second well due to the free drug.

TABLE 2 Representative portion of the screening of the plates Culturewell OD in absence of free drug OD in presence of free drug 1 H12 3.5680.504 37F5 0.738 0.358 2B11 3.942 3.649 19D5 1.152 0.225 24D11 3.3051.342

Subsequent to the selection of a clone from the fusion culture plates,the cells were subjected to stringent cloning via limiting dilution.Subclones growing from those wells in which single cells had beenverified by microscopy were then re-tested by the above method.Stability of antibody expression was judged on the number of wellsshowing antibody, the level of binding and the presence of any wellsshowing growth but little or no antibody. If any of the latter werefound, a well showing high antibody secretion was then used to repeatstringent subcloning. This was repeated as necessary to obtain 100% ofthe subclones secreting equivalent quantities of antibody. Cells fromselected wells were then expanded in culture, and used to preparepreliminary cell banks. The supernatant from those cultures was thensubjected to specificity analysis.

The antibody containing culture supernatants from the expansion cultureswere subjected to specificity analysis by the following procedure.First, the titer appropriate for analysis was determined by dilutionanalysis. A dilution of antibody providing for approximately 50% ofmaximal binding was selected for proceeding to the next step. Second,binding to the saquinavir-BSA conjugate was examined at the aboveantibody dilution, in the presence of varying amounts of six HIVprotease inhibitor drugs. The data was subjected to analysis bynon-linear regression curve fitting to a 4-parameter logistic function.That parameter which describes the concentration of the free drug whichcorresponds to 50% of the binding in the absence of free drug is termedthe ED₅₀ for that drug. The specificity of the antibody can thus bedescribed by comparing the ED₅₀ of the cognate drug, saquinavir, or saqED₅₀ with the other values for other drugs fitted from those dataaccording to the following equation (using nelfinavir data for thisexample):

${\%\mspace{14mu}{cross}\text{-}{reactivity}} = {\frac{{saq}\;{ED}_{50}}{{nel}\;{ED}_{50}} \times 100.}$

The four parameter logistic function used is

${ODx} = {\frac{{OD}\;\max}{\left( {1 + \left( \frac{{ED}_{50}}{X} \right)^{S}} \right.} - {{OD}\;\min}}$where S is the curvature parameter, ODmax is the optical density with 0drug concentration, ODmin is the optical density of the background ofthe instrument, and ODx is the optical density observed at drugconcentration X in moles/liter (M/l).

By this analysis, the cross-reactivities of two anti-saquinavirantibodies are given in Table 3. Murine hybridomas SAQ 10.2.1 and SAQ14.1.1 were deposited with the American Type Culture Collection (ATCC)on Jan. 18, 2002 and assigned ATCC No. PTA-3973 and ATCC No. PTA-3974,respectively.

TABLE 3 Specificity of saquinavir 10.2.1 and 14.1.1 antibodies CloneSaquinavir Nelfinavir Indinavir Amprenavir Ritonavir Lopinavir 14.1.1 %Cross 100 0.003 0.100 0.053 0.134 0.075 Rx ED₅₀ (M/l) 4.9 × 10⁻⁸ 1.72 ×10⁻³ 4.9 × 10⁻⁵ 9.35 10⁻⁵ 3.66 10⁻⁵ 6.52 10⁻⁵ 10.2.1 % Cross 100 0   0    0    0    0    Rx ED₅₀ (M/l) 1.7E⁻⁸ <1E⁻⁴ <1E⁻⁴ <1E⁻⁴ <1E⁻⁴ <1E⁻⁴

EXAMPLE 79 Development of Monoclonal Antibodies to Nelfinavir

The procedures used for the development of monoclonals to nelfinavirwere similar to those used for saquinavir. Female Balb/c mice 8 weeks ofage, were immunized with 100 μg of conjugate 5F emulsified in CompleteFreund's Adjuvant via intraperitoneal injection. Twenty one days later,another immunization of the same dose followed in Incomplete Freund'sadjuvant. Four further injections were carried out, using the samedosage and alternating with Ribi adjuvant, at approximately 21 dayintervals. All adjuvants were from the Sigma Chemical Co.

Four days following the last injection, a mouse was killed byexsanguination and cervical dislocation. Spleen cells were taken andfused to the F0 myeloma line by the same procedure as for saquinavir.Culturing and feeding were also the same.

Screening of growing hybridomas was as for saquinavir, with theexception that nelfinavir-BSA (5G) and free nelfinavir were substitutedfor the saquinavir-BSA and free saquinavir, respectively. Table 4presents a portion of the screening data thus obtained.

TABLE 4 Development of nelfinavir clones Culture well OD w/o freenelfinavir OD with free nelfinavir 9 D4 4.200 1.812 56 A9 3.906 0.469 12G3 3.948 2.482 46 B12 3.946 1.869 12 A6 3.955 0.456 40 E7 3.820 0.271

Further processing to assure stability was by the same methods as forsaquinavir monoclonal antibodies. Specificity analysis was using thesame panel of drugs, with competitive binding by nelfinavir taken as100%. Table 5 shows the specificities of subclones of the lines shown inTable 4.

TABLE 5 Specificities of selected stabilized subclones of nelfinavirclones Clone Saquinavir Nelfinavir Indinavir Amprenavir RitonavirLopinavir 5.4.1 % Cross 0 100 0 0 0 0 Rx ED₅₀ (M/l)  >4 × 10⁻⁴ 1.1 ×10⁻⁹ >4 × 10⁻⁴ >4 × 10⁻⁴ >4 × 10⁻⁴ >4 × 10⁻⁴ 15.3.1 % Cross 0 100 0 0 00 Rx ED₅₀ (M/l)  >4 × 10⁻⁴ 2.4 × 10⁻⁸ >4 × 10⁻⁴ >4 × 10⁻⁴ >4 × 10⁻⁴ >4 ×10⁻⁴ 21.4 % Cross    0.041 100    0.033    0.018 0   0.07 Rx ED₅₀ (M/l)1.3 × 10⁻⁶   5.3 × 10⁻¹⁰ 1.6 × 10⁻⁶  2.9 × 10⁻⁶  >4 × 10⁻⁴ 7.7 × 10⁻⁸ 

Murine hybridoma NEL 5.4.1 was deposited with the American Type CultureCollection (ATCC) on Jun. 25, 2002 and assigned ATCC No. PTA-4475.

EXAMPLE 80 Development of Monoclonal Antibodies to Indinavir

12-week old female Balb/c mice were given a primary intraperitonealimmunization with 100 μg indinavir KLH conjugate 4G together with theadjuvant CFA (complete Freund's adjuvant). This was followed by threefurther intraperitoneal immunizations after 6 weeks at monthlyintervals. In this case each mouse was administered with 100 μgindinavir KLH conjugate 4G together with IFA (incomplete Freund'sadjuvant). Subsequently the last immunizations were carried outintravenously with 100 μg indinavir KLH conjugate 4G in PBS buffer onthe second day and on the last day before fusion.

The spleen cells of the mice immunized as described above were fusedwith myeloma cells according to Galfré, Methods in Enzymology, Vol. 73,3 (1981). Approximately 1×10⁸ spleen cells of the immunized mouse weremixed with 2×10⁷ myeloma cells (P3X63-Ag8-653, ATCC CRL 1580) andcentrifuged (10 minutes at 300 G and room temperature). The cells werethen washed once with RPMI 1640 medium without fetal calf serum (FCS)and again centrifuged at 400 G in a 50 mL conical tube. Subsequently 1mL PEG (polyethylene glycol, molecular weight 4000, Merck, Darmstadt)was added, and it was mixed by gentle shaking. After 1 minute in a waterbath at 37° C., 5 mL RPMI 1640 without FCS were added dropwise, mixed,made up to 30 mL with medium (RPMI 1640) and subsequently centrifuged.The sedimented cells were taken up in RPMI 1640 medium containing 10%FCS and plated in hypoxanthine-azaserine selection medium (100 mmol/lhypoxanthine, 1 μg/mL azaserine in RPMI 1640+10% FCS). Interleukin 6from mouse (Roche Diagnostics GmbH, Catalog No. 1 444 581, 50 U/ml) wasadded to the medium as a growth factor.

After approximately 11 days the primary cultures were tested forspecific antibody synthesis. Primary cultures which exhibited a positivereaction with indinavir and no cross-reaction with saquinavir,nelfinavir, ritonavir and amprenavir, were cloned in 96-well cellculture plates by means of a cell sorter.

The deposited cell lines/clones listed in Table 6 were obtained in thismanner. Murine hybridomas <INDIN>M 1.003.12 and <INDIN>M 1.158.8 weredeposited with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ) on Jun. 18, 2002 and assigned DSM No. ACC2547and DSM No. ACC2546, respectively.

TABLE 6 Immunoglobulin subclass Clone IgG subclass 1.158.8 IgG1 kappa1.003.12 IgG2a kappa

For determination of the specificity of the antibodies in the culturesupernatant of the hybridoma cells, microtiter plates coated withrecombinant streptavidin (MicroCoat Co. Penzberg, Catalog No. 148051001)were coated with 500 ng/mL of indinavir biotin conjugate 4I (100 μL perwell diluted in PBS/1.0% CROTEIN C/0.1% TWEEN 20; incubation overnightat 4° C.) and subsequently washed 3 times with 0.9% NaCl/0.1% TWEEN 20.(CROTEIN C is a trademark of Croda Colloids, Ltd. for hydrolyzedcollagen protein.)

Free streptavidin binding sites were then blocked by incubation with 100μg/mL of biotin (1 hour; ambient temperature while shaking) andsubsequently washed 3 times with 0.9% NaCl/0.1% TWEEN 20.

Next, 50 μL of the analyte to be tested for cross-reaction was added toa coated well in a concentration series of 0-25 μg/mL (diluted in PBSplus 1.0% CROTEIN C, 0.1% TWEEN 20) and together with 50 μL of theantibody solution (culture supernatant) to be examined and incubated for1 hour at room temperature while shaking. After washing 3 times with0.9% sodium chloride/0.1% TWEEN 20, 100 μL of a horseradishperoxidase-labeled Fab fragment of a polyclonal antibody from the sheepagainst mouse Fc (pab<mouse Fc gamma>S-Fab-POD, Roche; 25 mU/ml) wasadded to each well to detect bound antibody from the sample, incubatedfor 1 hour at room temperature while shaking and subsequently washed 3times with 0.9% sodium chloride/0.1% TWEEN 20.

Finally 100 μL/well ABTS solution (Roche Diagnostics GmbH, cat. no.1684302) was added and the absorbance at 405/492 nm was measured after30 minutes at room temperature in a SLT Spectra Image microplate readerfrom TECAN.

Using the test system described above, it was shown that the monoclonalantibodies <INDIN> M 1.158.8 and <INDIN> M 1.003.12 exhibited less than10% cross-reactivity with indinavir, nelfinavir, ritonavir, saquinavir,and amprenavir. FIG. 17 shows graphs of the cross-reaction of mab<INDIN> M 1.158.8 and mab <INDIN> M 1.003.12 with indinavir, nelfinavir,ritonavir, saquinavir and amprenavir.

EXAMPLE 81 Development of Monoclonal Antibodies to Amprenavir

Monoclonal antibodies to amprenavir were developed using the proceduresfor immunization, fusion, culture, and cloning as described above inExample 80.O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-amprenavirconjugate with KLH (3E) was used as the immunogen.

ELISA screening was accomplished using the amprenavir-biotin conjugate3J. Murine hybridoma <AMPREN> M 1.1.52 was deposited with the DSMZ onSep. 16, 2003 and assigned DSM No. ACC 2612.

Specificity was determined as described above in Example 80. It wasshown that the monoclonal antibody <AMPREN> M 1.1.52 exhibited less than10% cross-reactivity with indinavir, nelfinavir, ritonavir, saquinavir,and lopinavir. FIG. 20 shows a graph of the cross-reaction of mab<AMPREN> M 1.1.52 with indinavir, nelfinavir, ritonavir, saquinavir andlopinavir.

EXAMPLE 82 Development of Monoclonal Antibodies to Lopinavir

Monoclonal antibodies to lopinavir were developed using the proceduresfor immunization, fusion, culture, and cloning as described above inExample 80.O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-lopinavir conjugatewith KLH (6F) was used as the immunogen.

ELISA screening was accomplished using the lopinavir-biotin conjugate6G. Murine hybridoma <LOPIN> M 1.1.85 was deposited with the DSMZ onSep. 16, 2003 and assigned DSM No. ACC 2611.

Specificity was determined as described above in Example 80. It wasshown that the monoclonal antibody <LOPIN> M 1.1.85 exhibited less than10% cross-reactivity with indinavir, nelfinavir, ritonavir, saquinavir,and amprenavir. FIG. 21 shows a graph of the cross-reaction of mab<LOPIN> M 1.1.85 with indinavir, nelfinavir, ritonavir, saquinavir andamprenavir.

EXAMPLE 83 Development of Monoclonal Antibodies to Ritonavir

Monoclonal antibodies to ritonavir were developed using the proceduresfor immunization, fusion, culture, and cloning as described above inExample 80.O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-ritonavir conjugatewith LPH (1E) was used as the immunogen.

ELISA screening was accomplished using the ritonavir-biotin conjugate1D. Murine hybridoma <RITON> M 1.5.44 was deposited with the DSMZ onSep. 16, 2003 and assigned DSM No. ACC 2613.

Specificity was determined as described above in Example 80. It wasshown that the monoclonal antibody <RITON> M 1.5.44 exhibited less than10% cross-reactivity with indinavir, nelfinavir, lopinavir, saquinavir,and amprenavir. FIG. 22 shows a graph of the cross-reaction of mab<RITON> M 1.5.44 with indinavir, nelfinavir, lopinavir, saquinavir andamprenavir.

EXAMPLE 84 Synthesis of O^(c)-(N-FMOC-aminocaproyl)-atazanavir (7A)

O^(c)-(N-FMOC-aminocaproyl)-atazanavir (7A) was prepared by stirringatazanavir (7, 0.20 g), FMOC-aminocaproic acid (0.010 g, 1 eq), DCC(0.059 g, 1 eq), and DMAP (0.038 g, 1 eq) in dry methylene chloride (40mL) in a similar manner to Example 1, except that after stirringovernight at room temperature, an additional 0.5 eq of FMOC-aminocaproicacid and 0.5 eq of DCC were added, and stirring continued for a further3 days. Work-up and purification in a similar manner to that given inExample 1 gave the product 7A (210 mg; 71%) as a white solid. M+H 1040.5

EXAMPLE 85 Synthesis of O^(c)-(aminocaproyl)-atazanavir (7B)

O^(c)-(aminocaproyl)-atazanavir (7B) was prepared fromO^(c)-(N-FMOC-aminocaproyl)-atazanavir (7A) of Example 84 (0.092 g)following the conditions described in Example 9, except that two silicagel chromatography purifications were performed (first column using 40%methanol in ethyl acetate (EtOAc), second column using 20% methanol inEtOAc) to give the product 7B as a solid (0.070 g, 97%). M+H 818.4

In another run, 7B was isolated as the trifluoroacetic acid (TFA) saltafter purification by preparative RP-HPLC (C18, gradient of 5% to 100%of 0.1% TFA-acetonitrile in 0.1% TFA-water).

EXAMPLE 86 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir (7C)

O^(c)-(aminocaproyl)-atazanavir (7B) as the TFA salt (0.070 g),triethylamine (22 μL), and succinimido-oxycarbonyl butyryl chloride(0.0195 g) were stirred for 3 hours in dry THF at about 0° C. (ice-waterbath). The reaction was evaporated to dryness, redissolved in 15% THF inethyl acetate, and purified by silica gel chromatography (elution with30% THF in EtOAc, column pre-washed with several column volumes of 15%THF in EtOAc). Fractions containing product were combined, evaporated,redissolved in dry methylene chloride (CH₂C₁₂) and re-evaporated(repeated several times) to yieldO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir (7C) asa solid (24 mg, 31%). M+H 1029.4

EXAMPLE 87 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavir(7D)

A solution of O^(c)-(aminocaproyl)-atazanavir (7B, 0.054 g) in 2 mL ofdry DMF was added slowly to a stirring, cooled solution (ice-water bath)of disuccinimidyl terephthalate (0.0228 g) in 4.5 mL of dry DMF. Afterbrief stirring, triethylamine (50 μL) was added and the reaction stirredovernight. Analysis by HPLC indicated essential completion of thereaction. Solvent was removed on a rotovap under high vacuum (at lessthan 25° C.), the residue redissolved in acetonitrile-water and purifiedby preparative RP-HPLC (C18, gradient of 5% to 100% of 0.1% TFA-water)to give, from the main peak after evaporation of acetonitrile, freezingand lyophilization, the productO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavir(7D), assigned as the trifluoroacetic acid salt, in two cuts (0.036 gand 0.007 g, combined 0.043 g, 55%). M+H 1063.5 (free base)

EXAMPLE 88 Synthesis ofO^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavirConjugate with KLH (7E)

O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir KLHconjugate was prepared from purified keyhole limpet hemocyanin (60 mg)and O^(c)-(succinimido-oxycarbonyl-butyryl-aminocaproyl)-atazanavir (7C)from Example 86 (17 mg) following the general conditions described inExample 61, except that the reaction was performed in 40% DMSO. Proteinquantification of the retentate by Coomassie Blue Protein Assay showed10.8 mg/mL, 92% protein recovery (KLH standard/control). Aminequantification by TNBS colorimetric assay showed 56% lysinemodification.

EXAMPLE 89 Synthesis ofO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavirConjugate with BSA (7F)

O^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavir BSAconjugate was prepared from bovine serum albumin (100 mg) andO^(c)-[4′-(succinimido-oxycarbonyl)-benzoyl-aminocaproyl]-atazanavir(7D) as the TFA salt, from Example 87 (3 mg) following the generalconditions described in Example 61, except that the reaction wasperformed in 40% DMSO. Protein quantification by Coomassie Blue proteinassay showed quantitative recovery of protein at 10.0 mg/mL (BSAstandard/control). UV difference spectroscopy showed the ratio of haptento BSA to be 1:1.7

1. A compound having the structure


2. A compound having the structure


3. A compound having the structure

wherein KLH is keyhole limpet hemocyanin and n is a number from 1 to 50per 50 kilodaltons molecular weight of KLH.
 4. A compound having thestructure

wherein BSA is bovine serum albumin and n is a number from 1 to 50 per50 kilodaltons molecular weight of BSA.
 5. Murine hybridoma <INDIN> M1.003.12 having DSMZ No. ACC2547.
 6. Murine hybridoma <INDIN> M 1.158.8having DSMZ No. ACC2546.